Method and apparatus for determination of chemical species in perspiration

ABSTRACT

A dermal patch to be worn on the skin for increasing the concentration of an analyte expressed through the skin in perspiration to a conveniently measurable level. Included are patches and methods of using such patches to determine the quantity of an analyte in a given volume of perspiration, to determine a subject&#39;s sensitivity to an allergen, to allow an analyte to be detected with conventional detection systems, and to dissolve the structural components of a patch to facilitate analyte detection.

This is a division of application Ser. No. 07/989,204, filed Dec. 11,1992, now U.S. Pat. No. 5,441,048, which is a continuation-in-part ofapplication Ser. No. 07/569,007, filed Aug. 15, 1990, now U.S. Pat. No.5,203,327, which is a continuation-in-part of application Ser. No.07/241,707, filed Sep. 8, 1988, now U.S. Pat. No. 4,957,108.

FIELD OF THE INVENTION

The present invention relates to dermal patches for accumulating ananalyte expressed through the skin in perspiration and to methods ofusing such patches.

BACKGROUND OF THE INVENTION

A. Chemical Analysis of Body Fluids

The determination of a patient's physiological status is frequentlyassisted by a chemical analysis of a body fluid of that patient. Such ananalysis normally determines the existence and/or concentration of achemical species in the body fluid of the patient as an indication ofthat patient's condition. Increasingly, such analyses are being used todetermine the presence of a drug of addiction or a metabolite of such adrug in the body fluid of an individual.

Many analytes of interest can be detected in urine, which is readilyavailable from a subject and can be collected non-invasively. For thesereasons, the primary method for detecting drugs of abuse today is urineanalysis.

Blood, however, is also frequently analyzed for the presence of drugs ofaddiction as well as a wide variety of other analytes. Blood collectionis, however, inherently invasive, and carries the risk of infectionassociated with any invasive procedure. Blood testing must also beconducted at a physician's office or at another facility equipped toanalyze blood, which reduces the convenience of blood tests andincreases their cost. In addition, testing a blood sample can onlyreveal information about chemicals or metabolites that are present inthe blood of the subject at the time the sample is taken, and cannotdetect the presence of such analytes over a period of time.

Perspiration can also be collected in order to analyze a chemicalspecies present in the body. The non-invasive manner in which it can becollected renders perspiration suitable for use outside of a physician'soffice. In addition, a variety of molecules which are expressed inperspiration can be analyzed.

B. Diagnostic Kits for Collecting Perspiration

A variety of diagnostic kits for monitoring an analyte in sweat havebeen developed. For example, U.S. Pat. No. 3,552,929 to Fields, et al.discloses a band-aid-type test patch suited for determining the chlorideion concentration in perspiration as a method of diagnosing cysticfibrosis. The apparatus disclosed in Fields comprises an absorptivesweat collecting pad with an impermeable overlying layer for the purposeof preventing evaporation. When the absorptive pad is saturated, thepatch is removed from the skin and exposed to a series of stripsimpregnated with incremental quantities of silver chromate or silvernitrate, the color of which undergoes a well known change uponconversion to the chloride salt.

U.S. Pat. No. 4,706,676 to Peck discloses a dermal collection devicewhich comprises a binder to prevent reverse migration of an analyte, aliquid transfer medium which permits transfer of an analyte from thedermal surface to the binder, and an occlusive cover across the top ofthe liquid transfer medium and binder. Peck also discloses theapplication of such a dermal collection patch to detect variousenvironmental chemicals to which humans are exposed. After the dermalcollection device has been worn on a patient's skin for a period oftime, the patch is removed for analysis, which involves the chemicalseparation of the bound substance of interest from the binding reservoirand thereafter undertaking qualitative and/or quantitative measurementof the substance of interest by conventional laboratory techniques.

Another quantitative sweat collection patch is disclosed in U.S. Pat.No. 4,756,314 to Eckenhoff. This patch uses a diffusion rate-limitedmembrane as a means to maintain a constant flow of fluid into the patch.The patch comprises an impermeable outer boundary structure, and istherefore an occlusive patch.

However, prior art diagnostic test patches are generally only useful fordetermining the presence of analytes which are present in sweat inrelatively high concentrations, such as halide ions. In addition, theocclusive outer layer-type devices of the prior art are susceptible tothe problem of back diffusion of perspiration and/or the analytescontained therein, including changes in the skin's transportcharacteristics, both outward (Brebner, D. F., J. Physiol, 175: 295-302(1964)) and inward (Feldmann, R. J., Arch. Dermat., 91: 61-666(1965)).The maintenance of this aqueous state also fosters bacterialcolonization. Thus, there remains a need in many diverse applicationsfor an improved method and apparatus for the non-invasive determinationof the presence or concentration of an analyte in a body fluid such asperspiration.

SUMMARY OF THE INVENTION

In one aspect, the present invention comprises a dermal patch to be workon the skin of a subject mammal for quantitatively determining thepresence and amount of an analyte in that subject's perspiration. Thisdermal patch includes:

a fluid permeable support layer in fluid communication with thesubject's skin when the patch is worn on the subject's skin, wherein thesupport layer comprises a rate-limited structure that limits the rate ofdiffusion of perspiration through the support layer; and

an absorbent material in fluid communication with the support layer forcollecting non-aqueous components of perspiration which diffuse throughthe support layer, the support layer being located between the absorbentmaterial and the subject's skin when the patch is worn on the subject'sskin.

In a preferred embodiment, a gas permeable layer is located between theabsorbent material and the outside of the patch, wherein water and otherfluids expressed through the skin of the subject are permitted to escapethrough the gas permeable layer in their vapor phase to the outside ofthe patch. In order to attach a patch to the skin of a subject accordingto this aspect of the present invention, the gas permeable layer canfurther include an adhesive composition applied to the outer perimeterof the outer protective layer on the side of the outer protective layerin contact with the skin of the subject. The gas permeable layer canalso be used to form a pooling area between the gas permeable layer andthe subject's skin when the patch is worn on the subject's skin. Such apooling area is used to collect excess perspiration that is not diffusedacross the support layer. In one embodiment, the absorbent material isin fluid communication with the pooling area only through the supportlayer.

The absorbent material in this aspect of the present invention can bemade from various materials, including paper or cotton gauze. In oneembodiment, the collection of an analyte of interest from theperspiration of the subject in the absorbent material is improved byproviding the absorbent material with a specific binding partner for theanalyte. For example, the binding partner can be an antibody. In anotherpreferred embodiment, the absorbent material of the patch can bedissolved into a solution such that the dissolved material and solutiondo not interfere with the analysis of the analyte to be detected.

The rate-limited structure of the patch is advantageously a membrane,such as a polycarbonate microporous membrane or a membrane made fromnylon 6/6. In a preferred embodiment, the rate-limited structure limitsthe rate of diffusion of perspiration across the support layer to lessthan the insensible rate of perspiration through the skin of thesubject.

In another aspect of the present invention, a dermal patch to be worn onthe skin of a subject mammal for determining the presence of an analytein the subject's perspiration is disclosed, wherein the patch comprisesan absorbent material in fluid communication with the skin of thesubject for collecting non-aqueous components of perspiration whichdiffuse through the skin of the subject, and wherein the absorbentmaterial can be dissolved into a solution such that the dissolvedabsorbent material and solution do not interfere with the analysis ofthe components of perspiration. The absorbent material in this aspect ofthe present invention can be made from any of a number of dissolvablematerials, including protein, nylon 6/6, phenolic, polyurethane (TP),and polyester (PBT). Likewise, a number of solvents for dissolving suchabsorbent materials can be used, including acids and bases, theparticular solvent depending on the material to be dissolved. Forexample, if the absorbent material is polystyrene, the solvent can be,selected from the group consisting of chlorinated hydrocarbons,aromatic hydrocarbons, esters, ketones, essential oils of high terpenecontent, and turpentine. Examples of these solvents are cyclohexanone,dichloroethylene, and methylenedichloride. The absorbent material canalso be provided with a specific binding partner for an analyte presentin the perspiration of the subject. In one embodiment, the non-aqueouscomponents of perspiration to be detected include a drug of abuse suchas cocaine.

In another embodiment, a patch according to this aspect of the presentinvention can be provided with an allergenic material placed in fluidcommunication with the skin of the subject. Such a patch can be used todetermine whether a subject is allergic to that particular allergen. Thepatch in this aspect can additionally comprise a fluid permeable supportlayer in fluid communication with the subject's skin and located betweenthe absorbent material and the subject's skin. The support layer, in oneembodiment, can further comprise a rate-limited structure between theabsorbent material and the skin, wherein the structure allows thepassage of perspiration through the structure but at a rate lower thanthe insensible rate of perspiration through the skin of the subject. Therate-limited structure can also comprise a separate layer of material.

The patch in this aspect of the invention can additionally comprise agas permeable outer protective layer located between the absorbentmaterial and the outside of the patch. This layer can, in oneembodiment, further define a pooling area between the outer protectivelayer and the skin of the subject when the patch is on the subject'sskin, the absorbent material being in fluid communication with thepooling area only through the rate-limited structure.

In yet another aspect of the present invention, a dermal patch to beworn on the skin of a subject mammal is disclosed which can be used todetermine the sensitivity of the subject to an allergen. Such a patchcomprises:

an absorbent material in fluid communication with the skin of thesubject for collecting non-aqueous components of perspiration whichdiffuse through the skin of the subject; and

an allergen located proximate to the patch which is in fluidcommunication with the subject's skin when the patch is worn on thesubject's skin.

In a preferred embodiment, the patch further comprises a gas permeablelayer located between the absorbent material and the outside of thepatch, wherein water and other fluids expressed through the skin of thesubject are permitted to escape through the gas permeable layer in theirvapor phase to the outside of the patch. This patch can additionallycomprise a support layer in fluid communication with the skin of thesubject, the support layer being located between the absorbent materialand the skin of the subject when the patch is worn on the subject'sskin. An agent for increasing the permeability of capillaries in thedermis immediately beneath the patch can be placed in fluidcommunication with the subject's skin when the patch is worn on thesubject's skin, for example in the support layer or in the absorbentmaterial.

In one embodiment of this aspect of the present invention, the absorbentmaterial can be dissolved into a solution such that the dissolvedmaterial and solution do not interfere with the analysis of the desiredbody components. The absorbent material can also contain a specificbinding partner for the desired body components indicative ofsensitivity to an allergen. Such a specific binding partner can be, forexample, an antibody or an antigen. This aspect of the invention canadditionally comprise an outer protective layer, which advantageouslyincludes an adhesive composition applied to the outer perimeter of theouter protective layer on the side of the outer protective layer whichcontacts the skin of the subject, in order to attach the patch to theskin of the subject. This embodiment can also comprise a rate-limitedstructure between the absorbent material and the skin of the subjectwhen the patch is worn on the skin of the subject, as well as a poolingarea between the outer protective layer and the skin of the subject whenthe patch is on the subject's skin, the absorbent material being influid communication with the pooling area only through the rate-limitedstructure.

In a further aspect of the present invention, a method of quantitativelydetermining the presence of an analyte contained in the perspiration ofa subject mammal is disclosed, comprising the steps of:

a. placing a patch on the skin of a mammal, wherein the patch comprisesan absorbent material capable of concentrating non-aqueous components ofthe perspiration of the mammal;

b. passing perspiration of the mammal through a rate-limited structureat a known rate to the absorbent material, the structure beingpositioned between the skin of the mammal and the absorbent materialwhen the patch is worn on the skin of the mammal, the structure being ofa known area;

c. removing the patch after a sufficient test period of time has elapsedso that the analyte can be detected by an assay for the analyte;

d. recording the amount of time the patch was worn in order to determinethe total amount of perspiration which passed across the structure;

e. determining the amount of an analyte contained in the patch; and

f. relating the amount of analyte determined in step (e) to the amountof perspiration determined in step (d) in order to determine the averageamount of the analyte in the mammal's perspiration.

The rate-limited structure in this aspect of the invention canadvantageously be either a polycarbonate microporous membrane or amembrane made from nylon 6/6. The absorbent material, in one embodiment,can also be paper. The absorbent material in this aspect can alsoadvantageously have attached thereto a binding partner for a specificanalyte. For example, the binding partner can be an antibody or anantigen.

In yet a further aspect of the present invention, a method of detectingmetabolites of an analyte contained in the perspiration of a subjectmammal is disclosed, the method comprising:

a) passing an analyte through the skin of the mammal in the perspirationof the mammal;

b) collecting the analyte in an absorbent material;

c) chemically modifying the analyte after the analyte has been collectedon the absorbent material; and

d) detecting the analyte.

In a preferred embodiment, this method can additionally comprise thestep of freeing the analyte from the absorbent material after theanalyte has been collected on the absorbent material. In thisembodiment, the freeing step can comprise eluting the analyte from theabsorbent material with a solvent. The freeing step can also comprisedissolving the absorbent material into a solution with a solvent suchthat the dissolved material and solution do not interfere with thedetection of the analyte, where the absorbent material comprises adissolvable material. In another preferred embodiment, the step ofchemically modifying the analyte comprises exposing the analyte to asolution having an alkaline pH. This method can also further compriseheating the analyte during the step of chemically modifying it. In yetanother embodiment, this step can comprise incubating the analyte withan enzyme capable of hydrolyzing the analyte. In all of the foregoingembodiments, any of a number of analytes can be detected, such ascocaine.

A further aspect of the present invention is a method of determiningwhether a subject mammal is allergic to a particular allergen. Thismethod comprises:

placing, a patch on the surface of the skin of the mammal, wherein thepatch comprises an absorbent material in fluid communication with theskin of the mammal when the patch is worn on the skin of the mammal, theabsorbent material including the allergen;

inducing the migration to the absorbent material of components of thebody of the mammal associated with an allergic reaction to the allergen;and

detecting the components to determine whether the mammal has expressedan allergic response to the allergen.

In one embodiment of this aspect of the present invention, the absorbentmaterial additionally comprises a composition for increasing thepermeability of capillaries in the dermis immediately beneath the patch.

In yet another aspect of the present invention, a method for determiningwhether a subject mammal is allergic to a particular allergen isdisclosed which comprises the steps of:

exposing the skin of the mammal to the allergen;

accumulating perspiration from the mammal proximate to the area of theskin of the mammal exposed to the allergen; and

detecting the presence of an analyte in the perspiration, wherein theanalyte is indicative of an allergic reaction to the allergen.

In one embodiment, this method further comprises applying a compositionto the skin of the mammal that increases capillary permeability.

Another aspect of the present invention comprises a method fordetermining the presence of an analyte in the perspiration of a subjectmammal, comprising the steps of:

accumulating perspiration containing an analyte from the subject on anabsorbent material, wherein the absorbent material can be dissolved by asolvent into a solution;

dissolving the absorbent material containing the analyte with a solvent,wherein the solvent does not interfere with the detection of theanalyte;

detecting the analyte in the solution.

In one embodiment, the detecting step additionally comprises chemicallymodifying the analyte and detecting a metabolite of the analyte in orderto detect the presence of the analyte. In a preferred embodiment, theabsorbent material is a material selected from the group consisting ofprotein, nylon 6/6, phenolic, polyurethanel (TP), and polyester (PBT).In this embodiment, the solvent is preferably selected from the groupconsisting of an acid and a base. In a further embodiment, the absorbentmaterial is polystyrene and the solvent is selected from the groupconsisting of chlorinated hydrocarbons, aromatic hydrocarbons, esters,ketones, essential oils of high terpene content, and turpentine. Forexample, the solvent can be cyclohexanone, dichloroethylene, ormethylenedichloride.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of a dermal patch according to oneembodiment of the present invention.

FIG. 1a is a cross-sectional view along the line 1a-1a of the dermalpatch of FIG. 1.

FIG. 2. is a perspective view of a dermal patch according to a secondembodiment of the present invention.

FIG. 2a is a cross-sectional view along the line 2a-2a of the dermalpatch of FIG. 2.

FIG. 3 is a perspective view of a third embodiment of the dermal patchof the present invention.

FIG. 3a is a cross-sectional view along the line 3a-3a of the patch ofFIG. 3.

FIG. 4 is a perspective view of one embodiment of a reagent packet foruse in effecting a color change responsive to the presence of analyte inthe patch of the present invention.

FIG. 5 is an exploded cross-sectional schematic view of a fourthembodiment of the present invention.

FIG. 6 is a cross-sectional view of a dermal patch according to afurther embodiment of the present invention.

FIG. 7 is a perspective view of a dermal patch according to anotherembodiment of the present invention.

FIG. 8 is an exploded elevational view of a dermal patch according toyet another embodiment of the present invention.

FIG. 9 is a plan view of a dermal patch according to a furtherembodiment of the present invention.

FIG. 10 is an exploded elevational view according to still anotherembodiment of the present invention.

FIG. 11 is a cross-sectional view of a dermal patch of the presentinvention which includes a pooling area.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

I. Dermal Patches for Detecting Analytes

A. Non-Occlusive Dermal Patches

Referring to FIG. 1, there is disclosed a dermal patch 10 according toone embodiment of the present invention, illustrated as being secured tothe surface of the skin 12 of a subject. As will be appreciated by oneof skill in the art, the patch of the present invention may be used forveterinary purposes as well as on humans. In addition, the patch can beused in more diverse applications such as in agriculture or any otherenvironment where a chemical species is to be detected in a fluid. Thepreferred use, however, is for determination of preselected chemicalspecies or analyte in sweat (perspiration), and the ensuing discussionis principally directed to that use.

Moisture expressed from the skin 12 within the perimeter of the testpatch 10 first accumulates in a concentration zone 14 beneath the firstside of a gas permeable filter or layer 16 which is in fluidcommunication with the skin 12. The concentration zone 14 preferablycontains an absorbent material, such as a fluid permeable medium 20which may be cotton gauze or other commonly available fluid permeablematerial. For example, a layer of any of a variety of known fiber webssuch as knitted fabrics, or non-woven rayon or cellulose fibers may beused. Filtration Sciences #39 is a particularly preferredfluid-permeable medium for use as a concentration zone in the presentinvention. In a preferred embodiment, the absorbent material containsbinders, such as antibodies, for specifically binding analytes ofinterest to the absorbent material of the patch. As used herein, theterm "absorbent material" designates any fluid permeable materialcapable of collecting or holding analytes contained in perspiration.Preferably, such a material is also able to concentrate such analytes onthe patch.

The term "fluid permeable" is used herein to describe a material whichwill permit the passage of the liquid phase of fluids expressed from theskin and which will also allow the passage of the vapor phase of suchfluids. A fluid permeable filter or layer will thus allow the passage ofwater in both the liquid and vapor phases. "Water" is used herein todenote both the liquid and vapor phases of water unless reference isspecifically made to a particular phase.

Moisture from perspiration accumulates in the interfiber spaces of themedium 20. Under the influence of body heat which is readily conductedfrom the surface of the skin through the liquid phase, the liquid watercomponent of the perspiration will tend to volatilize. Such volatilizedwater can thereby pass through the gas permeable filter or layer 16,which is located on the side of the medium 20 distal of the skin 12, andleave the patch 10.

As previously discussed, the patch 10 is provided with a gas permeablefilter 16. The term "gas permeable" is used to describe a material whichpermits the passage of gases, including the vapor phase of fluidsexpressed from the skin, but substantially retains the fluid phasewithin the patch. Any of a variety of suitable commercially availablemicrofiltration membrane filters may be used for this purpose, such as.the Gore-Tex 0.45 micron Teflon filter manufactured by W. L. Gore &Associates, Inc. (Elkton, Md.).

Adjacent a second side of the gas permeable filter 16 is a dischargezone 18. As previously discussed, the gas permeable filter 16 retainsthe fluid phase but permits escape of the vapor phase of the fluidcomponent in perspiration. Thus, the vapor component, which primarilyconsists of vaporized water, continuously escapes through the gaspermeable filter 16 exiting the second side thereof into discharge zone18, which is in communication with the atmosphere. In an alternativeembodiment, not separately illustrated, the gas permeable filter 16 isreplaced by a fluid permeable membrane which permits passage of theliquid phase. In this embodiment, liquid, or a combination of vapor andliquid, will be permitted to escape from the patch. Any of a variety offluid permeable filters are commercially available which can be used toform a fluid permeable filter used in this embodiment of the presentinvention. A preferred fluid permeable filter is constructed from JamesRiver Paper Drape.

A flexible, gas permeable outer layer 22 is preferably disposed adjacentthe second side of filter 16 in the discharge zone 18. This layer servesto protect the filter 16 against physical damage such as abrasion, andcan also serve as a barrier for preventing chemical contamination of thefilter material from the outside. Outer layer 22 may comprise any of avariety of commercially available gas permeable tapes and films whichare known to one of skill in the art. Outer layer 22 may also bedistinct from or integral with tape 26, discussed below. Alternatively,depending upon the intended application of the patch, outer layer 22 maybe deleted altogether, where it does not appear that abrasion orexternal contamination will deleteriously affect the patch 10, or wherethe gas permeable layer 16 is made from a material which is itselfresistant to abrasion and/or external contamination, thus obviating theneed for the outer layer 23.

The patch 10 illustrated in FIG. 1 is secured to the surface of the skinby means of a peripheral band of tape 26. Preferably, the tape 26 willextend around all sides of the patch 10. For example, an annular ring oftape can be die punched for use with a circular patch, or the center ofa rectangular piece of tape can be removed to expose outer layer 22 orfilter 16 of a rectangular patch (see FIGS. 1 and 3, respectively).Alternatively, outer layer 22 and tape 26 can be deleted altogether andlayers 16 and 20 can be secured to the surface of the skin by a bandageor through the use of an adhesive. One such method would be to capturelayers 16 and 20 under a bandage or wrapping surrounding the arm or theleg. In this case, the gases and/or fluids are permitted to escapethrough layers 16 and 20 and into the bandage, where they may eithercollect or from which they are dissipated into the environment.

A large variety of hypoallergenic or other suitable tapes are well knownin the art, which may be adapted for use with the patch 10 of thepresent invention. Different tapes or adhesives may be desirabledepending upon the intended use of the test kit, based upon theirability to adhere to the skin during different conditions such asdaytime wearing under clothing, during sleep, during profuse sweatingfor prolonged periods or during showers. It has been determined that themost desirable tapes include multiple perforations which prevent sweatfrom building up underneath the tape and eventually compromising theintegrity of the adhesive. Preferably, a tape, such as Dermiclearmarketed by Johnson & Johnson, is used. More preferably, the tapecomprises a layer of 3M 1625 Tegaderm wound dressing available from the3M Company (St. Paul, Minn.).

Any of a wide variety of means for securing the patch 10 to the skin 12may be utilized. For example, the tape 26 can be eliminated and gauzelayer 20 provided with a lower adhesive layer to perform the samefunction. One such adhesive membrane is the MN-100 adhesive membranemanufactured by Memtec of Minnetonka, Minn. This membrane is fluidpermeable so that it passes fluid as would the gauze layer 20, yet hasone adhesive side so that it will stick to the skin. Alternatively,outer protective layer 22 can comprise an annular flange 23, extendingradially outwardly beyond the outer edges of filter 16 and gauze 20 (seeFIG. 2a). The lower surface of the flange 23 is then provided with asuitable adhesive.

The surface temperature of human skin varies regionally. However, it isgenerally within the range of from about 86° F. to about 90° F. at rest,and can rise to much higher temperatures under conditions of strenuousexertion. At those temperatures, a number of chemical species ofinterest for the purpose of the present invention, such as creatinekinase or a high or low density lipoprotein, have a sufficiently lowvapor pressure that volatilization is not a significant factor in theefficiency of the concentration function. At the same time, thesubstantial aqueous component will have a sufficiently high vaporpressure that it will tend to volatilize, thereby concentrating the lessvolatile fractions. However, in some applications the chemical speciesof interest will have a high enough vapor pressure, even at the restingtemperature of human skin or the temperature of another surface to whicha patch of the present invention is applied, such that escape of thevapor phase through the gas permeable filter 16 of the analyte ofinterest will disadvantageously impair the efficacy of the test patch.For these analytes, a modified patch must be used.

B. Dermal Patches for Detecting Volatile Analytes

Referring to FIGS. 2 and 2a, there is disclosed a modified patch 11according to the present invention for use in detecting an analytehaving a propensity to escape through the gas permeable filter 16 as avapor under ordinary use conditions. The test patch 11 comprises aconcentration zone 14 defined on its inner boundary by the skin 12 towhich the patch 11 is secured. The outer boundary of the concentrationzone 14 is defined by the gas permeable filter or layer 16, whichseparates the concentration zone 14 from the discharge zone 18. Disposedin the concentration zone 14, and adjacent the gas permeable filter 16,is a binder layer 31 for binding and preventing the escape of moleculesof the volatile analyte. The binder layer 31 is separated from the gauzelayer 20 by a porous layer 28, which may comprise any of a variety offilms for retaining the binder layer 31 yet permitting passage of fluid.

In the embodiment illustrated in FIG. 2a, perspiration will pool in theinterfiber spaces of the gauze 20, and will percolate through porouslayer 28 into the binder layer 31. In that layer, a chemically active orbiochemically active binder material will act to selectively bindanalyte, there analyte, thereby preventing it from escaping as a vaporthrough gas permeable filter 16. As discussed in connection with theembodiment illustrated in FIG. 1, it is also possible to replace the gaspermeable filter 16 with a fluid permeable layer, where the presence offluid on the outside of the test patch would not be undesirable.

The binder layer 31 may comprise any of a variety of binders dependingupon the nature of the volatile analyte to be determined. For example,the binder may chemically bind with the analyte or adsorb the analyte tobe determined. Thus, when the analyte being collected is ethanol, thebinder layer advantageously contains activated charcoal. In addition,the binder layer may comprise a specific binding partner of the analyteto be determined, such as a polyclonal or monoclonal antibody or anantigen matched to a specific antibody desired to be measured in theperspiration.

The patch 11 is additionally provided with tape 26 or another means forsecuring the patch to the skin of a subject, as has. been detailed inconnection with the embodiment illustrated in FIG. 1. Patch 11 isillustrated, however, as having a unitary outer layer 22 extendingbeyond the perimeter of the underlying layers to form an annular flange23, which is provided with an adhesive on its lower surface. Asdiscussed in connection with the embodiment of FIG. 1, outer protectivelayer 22 permits the escape of water vapor yet protects the filtermaterial from chemical contamination from the outside. As also discussedabove, gas permeable layer 16 can also in some cases function as theouter layer 22.

C. Dermal Patches Having a Microbead Layer

Referring to FIGS. 3 and 3a, there is disclosed a further embodiment ofthe test patch of the present invention wherein an inner porous layer 28and an outer porous layer 30 define a space for containing a microbeadlayer 32. The microbeads of such a microbead layer 32 can desirably haveattached thereto a capture reagent, such as antibodies or other meansfor binding analytes of interest. The inner layer 28 and outer layer 30preferably comprise the same material, which may be any suitablematerial for providing an unrestricted flow of fluid through the patchwhile trapping the microbeads in between. One suitable material forporous layers 28, 30 is the fluid permeable and microporous film knownby the name Ultipor (nylon 6) and manufactured by Pall Corporation inGlen Cove, N.Y. Additional manufacturers of suitable nylon filtrationmembranes include Micron Separations, Inc. of Westborough, Mass., andCuno of Meridan, Conn. Porous layers 28, 30 may also be comprised ofmaterials other than nylon, such as polycarbonate, modifiedpolyvinylchloride and polysulfone.

The gauze, the inner and outer porous layers and the adhesive tape inall embodiments can be cut to size with conventional dies. The gauze 20and the inner porous layer 28 can be fixed to the adhesive ring 26 withconventional adhesives, such as those used on the adhesive surfaceitself. Alternatively, they could be heated or ultrasonically bondedtogether. The proper amount of microbeads can then be placed on top ofthe inner porous layer, after which the outer porous surface is attachedby similar means. Typically, in a one-inch diameter patch, from about0.05 grams to about 1 gram of microbeads will be used, and preferablyfrom about 0.1 to about 0.4 grams will be used. The inner and outerporous surfaces may have to be staked or spot-welded together in somepattern, as will be appreciated by one of skill in the art, to preventthe microbeads from collecting in one area.

The free adhesive surface is optimally covered by pull-away paper (notillustrated) with adequate space to be gripped with one's fingers. Thepatch is packaged in a paper or plastic pouch similar to that used inconventional bandaid packaging. The assembled unit could be terminallysterilized or pasteurized prior to sale. Alternatively, the packagecould comprise a vapor barrier such as a metallic foil or mylar and eveninclude oxygen or moisture absorbent means such as a small packet of anyof a variety of known desiccants, such as silica gel, calcium chloride,calcium carbonate, phosphorous pentoxide or others as will beappreciated by one of skill in the art.

The total thickness of microbead layer 32 can be varied considerably.However, if a color change is to be used to detect an analyte and thesuch color change is to be brought about by immersing the patch inappropriate reagent baths, layer 32 is preferably no more than about 3mm thick since color changes occurring at immobilized sites on thickerlayers would not likely be observable. More preferably, the microbeadlayer is between about 1 mm and about 2 mm thick. If such color changeanalysis is not performed, the microbead layer 32 can alternatively betorn open, releasing loose microbeads which can be used to conductchemical analysis for detecting the presence of an analyte bound to themicrobeads by conventional means, such as in a cuvette.

Optimally, the diameter of the beads in microbead layer 32 will be atleast about one order of magnitude larger than the diameter of the poresin inner porous layer 28 and outer porous layer 30. For example, thebeads contained in microbead layer 32 may have diameters within therange of from about 5 to 50 microns, and preferably within the range offrom about 5 to about 10 microns. If 10-micron diameter beads areutilized in the microbead layer 32, for example, inner porous layer 28and outer porous layer 30 will optimally comprise a median pore size ofapproximately 1 micron.

The microbead layer 32 may comprise any of a variety of known materialsincluding polystyrene, latex, and glass. Beads sized from approximately0.05 micron to 100 micron which are suitable for the present applicationare available from Polysciences of Warrington, Pa.

Microbead layer 32 serves as the support for an immobilized specificbinding partner for the analyte to be determined. Thus, a molecule witha high chemical affinity for a specific component in the fluid to beanalyzed will be immobilized to the microbeads in microbead layer 32.

D. Dermal Patches Having an Impermeable Outer Layer

Referring to FIG. 5, there is disclosed a further embodiment of thepresent invention, particularly suited for use under conditions in whichprofuse sweating is not present, such as in passive insensibleperspiration, wherein the test patch is provided with an impermeableouter layer 42. In order to minimize any back diffusion of fluid intothe skin, an absorptive layer 44 is provided to form a reservoir fordrawing moisture away from the surface of the skin and through support46 to which is bound a specific binding partner for at least one analyteto be determined. Layer 44 may include chemical means for binding orcollecting moisture such as a desiccant, as has been previouslydiscussed, which is suitable for use in proximity to the skin. The patchmay be further provided with an underlying porous layer 48 to separatesupport 46 from the surface of the skin, and the patch is provided withany of the means for attachment to the skin as have been previouslydiscussed.

E. Dermal Patches which Minimize Lateral Diffusion of Sweat in a Patch

Referring to FIG. 6, there is disclosed a modified patch 13 according tothe present invention, in which all intervening layers between the skin12 and the binder layer 30 have been deleted. By disposing the binderlayer (i.e., the layer having a specific binding partner for an analyteto be determined) directly adjacent the skin, lateral diffusion of sweatthroughout the binder layer 31 is minimized. The proximity of the binderlayer 31 to the 5 skin 12 allows the output of each duct of the sweatglands to contact or be in fluid communication with a relatively smallarea of the binder layer 31. For a variety of reasons which will beapparent to one of skill in the art, it may also be desired to mount amicroporous membrane, preferably a fluid permeable membrane 50 atop thebinder layer 31.

The evaporative capacity of the binder layer 30 and the fluid permeablemembrane 50 is preferably sufficient relative to the output capacity ofthe individual sweat ducts, to minimize lateral diffusion of sweat awayfrom the immediate area of the duct. This embodiment has specialapplication for monitoring the chemical composition of insensibleperspiration and/or non-exercise perspiration, in instances where outputfrom the sweat glands is limited. Due to the magnification effectdetailed infra, the present embodiment is also particularly suited formonitoring low concentration analytes.

By limiting the suppressive characteristics of moisture or water on theskin, through the use of materials having a maximal evaporativecapacity, the instant embodiment allows increase of the through-put rateof sweat in the patch by maximizing sweat gland output. Nadel andStolwijk (J. Applied Physiology, 35(5): 689-694 (1973)) disclose thatsweat gland activity is suppressed by water lying on the skin, finding adifference in whole body sweat rate of 40% between wet and dry skin.Mitchell and Hamilton (Biological Chemistry, 178: 345-361 (1948)), foundthat loss of water and solutes in insensible perspiration presumablystops whenever the surface of the skin is covered with a film of water.Brebner and Kerslake (J. Physiology, 175: 295-302 (1964)) postulate thatthe reason for this phenomenon is that water in contact with the skincauses the epidermal cells of the skin to swell and thus block the sweatducts.

The ability of the present invention to produce a positive responsebased upon the presence of relatively low concentrations of analyte isparticularly advantageous in view of the fact that, during activeexercise, a 1/4" diameter area of skin provides approximately 35microliters of sweat per hour, whereas a similar diameter area of skinproduces sweat at a non-exercise rate of only about 3.2 microliters perhour. The present embodiment is further advantageous as not requiringthe user to exercise, but only to wear the patch for an equal ortypically longer period during rest or at normal activity levels.

Thus, homogeneous diffusion of sweat throughout the binder layer ispreferably minimized when using the instant invention in conjunctionwith insensible and/or non-exercise perspiration and/or a determinationof minute amounts of analyte contained within perspiration. Theminimized lateral diffusion of perspiration throughout the binder layer31, according to the present invention, provides a more concentratedcollection of sweat at each sweat duct, thereby providing a greateramount of selected analyte to be determined at that area.

F. Dermal Patches Having Multiple Test Zones

Referring to FIG. 7, there is shown a modified binder layer 52 for apatch according to the present invention, wherein two or more distinctzones are provided on the binder layer 52. The use of a reference zoneor of several distinct test zones is contemplated for both the singlelayer patch discussed in connection with FIG. 6, as well as theembodiments discussed in connection with FIGS. 1-3a and 5. Themulti-zone binder layer 52 may also be used for certain embodiments tobe discussed hereinafter in connection with FIGS. 6-10 when specificbinding chemistry is used.

One or more of the zones, such as determination zone 60 (FIG. 7); isused to test for an analyte of interest within sweat, as detailedpreviously. One or more of the remaining zones, such as reference zone61, is used as a reference indicator.

Reference zone 61 performs a variety of functions, depending upon thedesired application of the test patch. For example, reference zone 61can be provided with color change chemistry as discussed previously toprovide the wearer with an indication that the patch has been worn forlong enough that a sufficient sample volume has traversed the patch toprovide a meaningful test for the analyte of choice. For this purpose,reference zone 61 is provided with affinity chemistry for a preselectedreference substituent such as IgG, albumin or any other sweat componentwhich is reliably present. Preferably, the selected referencesubstituent is one which provides a reasonably accurate measurement ofthe volume of sweat put through the system.

This use of the reference zone 61 may be facilitated by firstdetermining the rough concentration ratio of a reference substituentsuch as albumin to the analyte to be determined and providing the patchwith color change chemistry which provides a visual indication of thepresence of the reference substituent only well after the elution of theanalyte to be determined has exceeded the lower limits of detection.Reference substituents such as albumin will typically be present insignificantly greater quantities than the analyte. Thus, in order toaccomplish the objective of indicating passage of a sufficient samplevolume, the "sensitivity" of the patch for the reference substituent ispreferably lower than for the analyte. This can be achieved by using aproportionately lower amount of a specific binding partner for thereference substituent than for the analyte, other dilutions in theassay, or simply selecting a less abundant reference substituent.Selection of a suitable reference substituent and concentrationdeterminations can be readily made through simple experimentation by oneof skill in the art.

G. Use of Dermal Patches Having Multiple Test Zones to Prevent Tampering

Alternatively, and particularly useful in assays for drugs of abuse andtheir metabolites, a reference zone 61 (FIG. 7) can provide anindication that the skin patch was actually worn by the desired patient,parolee or other subject. One inherent limitation in a test in which asubject desires a negative result is the possibility that the subjectwill simply remove the patch after administration and replace it justprior to reexamination. This possibility gives rise to the ability ofthe wearer to ensure false negative results.

However, by provision of a reference zone 61 to detect a known componentin sweat, the test results will reveal test patches that have not beenworn for the test period. Reference zone 61 thus provides a method ofpreventing false negative evaluations due to tampering or removal of thetest patch.

A reference zone 61 to detect a known component in sweat may also beprovided as a positive control zone to ensure the discovery of falsenegative test results due to degradation of reagents or other componentsof the patch. In non drug-of-abuse screens, the indication producedwithin the reference zone 61 will preferably be a visible color changeby a chemical or antibody/antigen colorimetric interaction occurring orbecoming apparent to the wearer when a predetermined amount of thereference analyte has passed through the interaction area.

Optionally, a reference zone 61 may be provided as a negative control.zone to enable the discovery of false positive results. A preferrednegative control zone will have an immobilized specific binding partnerfor an analyte known to be absent in human sweat. The analyte's specificbinding partner must be known to not cross react with components presentin human sweat. An example of an appropriate analyte is bacteriophage T4coat protein.

In yet a further embodiment of the present invention (not illustrated)two or more analyte determination zones 60 are provided in a single testpatch. The use of multiple test zones is particularly useful inapplications such as a drug of abuse screen where testing for any one ormore of a wide variety of analytes may be desired. For example, a singletest patch may be used to screen for any of a plurality of drugs ofabuse, such as THC, Phencyclidine morphine and Methadone. A positiveresult for any of the drugs on the screen may provide sufficient proofof an offense such as a violation of parole, or can be used to signalthe need for more quantitative follow up investigations. Used as aninitial screening tool, the present invention offers the advantages ofbeing non-invasive, and much less expensive than conventionalquantitative analyses. For these reasons, a screening test patch asdisclosed herein is particularly suited for initial screening of largepopulations such as parolees, inmates, military personnel or otherswhere monitoring is desired.

The analyte determination zone 60 and analyte reference zone 61 may bephysically separated on the patch, such as in concentric circles ordiscrete zones, as illustrated in FIG. 7, or in the case of only two orthree analytes, interspersed throughout. In the latter case, positiveresults of different determinations would be indicated by the appearanceof different colors.

II. Placement of Dermal Patches

Although a patch of the present invention can be used to collectanalytes contained in any of a variety of body fluids, perspiration isthe desired fluid to be collected due to its dependable supply and itssimilarity to blood, albeit with lower analyte concentrations. Althoughcomponents found in saliva could also be collected with a patch of thepresent invention, saliva is often contaminated with molecules notexpressed by the body, such as foodstuffs. Therefore, in a preferredembodiment, the patches of the present invention are placed on the skinsurface of a subject.

A. Characteristics of Sweat Glands and Perspiration

Sweat glands are classified to be either of two types. Eccrine typesweat glands function primarily to regulate body temperature throughtheir relationship to evaporative heat loss. It is the eccrine typesweat gland that provides the sweat associated with exercise and istherefore the source of perspiration of interest for many applicationsof the patch of the present invention. Apocrine type sweat glands arelarger secreting elements which are localized only in relativelyisolated areas of the body such as the axilla, pubic and mammary areas.

Sato and Fusako (American J. Physiology, 245(2): 203-208 (1983))estimate that the diameter of the duct of the sweat gland isapproximately 40 microns. According to Scheupoein and Blank(Physiological Review, 51(4): 702-747 (1971)), the average density ofsweat glands on the skin surface is approximately 250 per squarecentimeter. Thus, the total surface area of sweat gland ducts of theskin represent 1/318 of the total surface area of the patch of theinstant invention. The visible result on a test patch of the presentinvention when, for example, using known ELISA technology to determine alow concentration analyte, is the appearance of a number of tiny colorchanges on the binder or absorptive layer associated with the output ofspecific ducts. If significant lateral diffusion of sweat is permittedprior to contact with the immobilized binding partner, the color changeis frequently too diffuse to detect with the naked eye.

Although the etiology of perspiration is relatively complex, it is knownto be caused by both mental states such as mental exercise and emotionalstress; thermal stress, as the sedentary body's response to temperaturecontrol; and exercise stress as the physically active body's response totemperature control.

In addition to the foregoing distinctions, perspiration can be eitherinsensible or sensible. Insensible sweat appears to be caused by waterdiffusion through dermal and epidermal layers. Its purpose appears to benot related to thermal regulation at all, but to aid in such things asthe improvement of mechanical interaction between the skin and surfacesto facilitate grip. Further complexities arise with regard to thespatial distribution of sweat glands and the flow rates of the varioustypes of perspiration. Specialized areas of the palms and soles of thefeet sweat continuously, although at a very low rate. The rate ofinsensible perspiration is dependent upon the position of the particulararea in question relative to the heart. For example, elevating a limbover the heart decreases the insensible perspiration rate in that limb.

At temperatures of about 31° C. in a resting human adult, insensibleperspiration proceeds at a rate of between about 6-10 grams per squaremeter per hour from the skin of the arm, leg and trunk, up to about 100grams per square meter per hour for palmer, planter and facial skin. Thelatter three areas jointly account for approximately 42% of the totalwater loss from the body due to insensible perspiration. Such insensibleperspiration first begins on the dorsal surfaces of the foot and spreadsto higher places on the body as the temperature increases. One reportedstudy determined that the average water loss due to insensibleperspiration for .a body surface area of 1.75 square meters ranged from381 ml, 526 ml and 695 ml per day at ambient temperatures of 22° C., 27°C. and 30° C., respectively.

In contrast to insensible perspiration which does not appear to beassociated with a particular surface element of the skin, sensibleperspiration has been associated with the eccrine gland. The number ofactively secreting eccrine glands varies among individuals and dependsupon the part of the body observed and the type of sweat responsecreated. Maximum gland density varies from between about 200 per squarecentimeter on the forearm to over 400 per square centimeter on the theireminence.

The appearance of sensible sweat begins at either when the skintemperature exceeds about 94° F. or the rectal temperature exceeds about0.2° F. over normal core temperature. Maximum rates of sweat volume losscan be as high as 2 liters per hour in average subjects and can be ashigh as 4 liters per hour for brief periods. Sensible perspirationbegins in the distal parts of the lower extremities and progressesupward as the environmental temperature is elevated. Thus, the dorsum ofthe foot begins to sweat long before the chest. The pattern of sensiblesweat response also shifts from one region of the body to another as thethermal stress increases. Under mild thermal stress, sweating is presentmainly in the lower extremities. As the thermal stress furtherincreases, sweating spreads to the trunk. Due to its large surface area,the trunk becomes the dominant water loss surface. Eventually, extremelyhigh rates are found in the trunk while rates in the lower extremitiesmay actually decline. The forehead can produce extremely high sweatrates but is among the last areas to sweat in response to thermalstress.

B. Placement of Dermal Patches

Although a patch of the present invention can be worn at any practicallocation on the body, preferable locations for the patch include theskin on the sole of the foot and areas on the chest, back, and biceps.The patch is able to be worn in confidence in these areas, and theseareas are not covered with excessive hair, so that the patch may besecured with conventional adhesive tapes.

The patch can advantageously be located on different regions of the bodydepending upon a variety of factors. It is well known that the quantityof perspiration generated is a function of both the location on thebody, as well as the physical activity during and immediately precedingcollection. This is due to both different densities of sweat glands ondifferent regions of the body, as well as to certain regulatoryfunctions of those glands.

Other desirable placement locations for the patches of the presentinvention will depend on the conditions under which it is desired todetect analytes. Using the parameters described above and other knownfactors, one of skill in the art will understand how to choose adesirable location on the body of a subject on which to place a patch.

III. Chemical Species Detectible with a Dermal Patch

A large variety of chemical species which are detectable in blood arealso present in sweat, although typically in a much lesserconcentration. Early investigation into the composition of perspirationcentered on electrolytes, including sodium, chloride, calcium andpotassium. Extreme individual variation was found among individuals, andthe electrolyte composition also differed depending upon whether thesweat was stimulated by thermal, mental or other etiology.

Further research has identified numerous additional components in sweat,including both electrolytes and more complex biological molecules. Someillustrative chemical species which have been identified in sweat areidentified in Table I below.

                  TABLE I                                                         ______________________________________                                        Chemical Components of Sweat                                                  ______________________________________                                        diphtheria antitoxin sulfates                                                 ascorbic acid        iodine                                                   thiamine             iron                                                     riboflavin           fluorine                                                 nicotinic acid       bromine                                                  amino acids          bismuth                                                  ethanol              lactic acid                                              antipyrine           pyruvate glucose                                         creatinine           nitrogen                                                 C-14 methylurea      ammonia                                                  C-14 acetamide       uric acid                                                C-14 urea            nicotine                                                 thiourea             morphine                                                 paraaminohippuric acid                                                                             sulfanilamide                                            mannitol sucrose     atabrin                                                  lactate              methadone                                                sodium chloride      phencyclidine                                            potassium            aminopyrine                                              calcium              sulfaguanidine                                           magnesium            sulfadiacine                                             phosphorous          amphetamines                                             manganese            benzoylecgonine                                          theophylline         phenobarbital                                            parathion            androgen steroids                                        tetrahydrocannabinol phencyclidine                                            insulin              phenytoin                                                cimetidine           carbamazepine                                            dimethylacetamide                                                             ______________________________________                                    

Any of the entries in Table I for which affinity chemistry can bedeveloped can be an appropriate subject of a test patch according to thepresent invention. Since most of the components listed in Table I arenon-volatile, they will be trapped in the concentration zone 14 of thepatch 10 illustrated in FIG. 1a, or on the binder layer 31 of FIG. 6.However, some components, most notably ethanol, would volatilize underthe influence of body heat, thereby enabling escape in the vapor phasethrough the test patch. Where the analyte to be determined is ethanol oranother volatile component, a patch of the present invention may bemodified as described in connection with the embodiment illustrated inFIG. 2 to contain specific binding partners for the analyte.

In one preferred embodiment, the analyte to be determined inperspiration is the enzyme creatine kinase MB (CK-MB) which is expressedfrom the cardiac muscle during myocardial infarction and other cardiacdistress. A monoclonal antibody raised against CK-MB can be immobilizedto the microbeads in accordance with any of a variety of conventionalmethods, such as the cyanogen bromide technique described in Pharmaciaproduct literature (Pharmacia, Inc., Piscataway, N.J.).

The antibody which is to be used for the purpose of complexing withCK-MB may be immobilized on any of a variety of supports known in theart. For example, anti-CK-MB antibody may be bound to polysaccharidepolymers using the process described in U.S. Pat. No. 3,645,852.Alternatively, the antibody may be bound to supports comprising filterpaper, or plastic beads made from polyethylene, polystyrene,polypropylene or other suitable material as desired. Preferably, thesupport will take the form of a multiplicity of microbeads which canconveniently be formed into microbead layer 32, illustrated in FIG. 3a.

As an alternative to a microbead support layer, the specific bindingpartner could be immobilized directly to the inner porous layer 20 or 28on FIG. 3a, to the underside of filter 16 of FIG. 1a, or to appropriateabsorbent materials used in any of the embodiments of the dermal patch.In this manner, the need for microbead layer 32 could be eliminatedentirely. Fluid permeable membranes which are specifically designed forbinding antibody proteins are commercially available, such as Zetaporfrom Cuno, and Protrans, available from ICN in Costa Mesa, Calif.

The monoclonal antibodies useful in the present invention can beproduced and isolated by processes which are well known in the art, suchas those discussed by Milstein and Kohler, reported in Nature, 256:495-497 (1975). In particular, Jackson describes a method of producinganti-CK-MM (an indicator of the status of skeletal muscles) andanti-CK-MB antibodies in Clin. Chem., 30/7: 1157-1162 (1984)).

Alternatively, the components of a commercially available diagnostic kitcan be utilized which incorporate the CK-MM enzyme chemically bound to abead support. A suitable kit marketed as the Isomune-Ck Diagnostic Kitby Roche of Nutley, N.J., is one commercially available candidate. Thiskit includes a goat antisera to human CK-MM and donkey anti-goatantibody covalently bound to styrene beads. A mixture would produce animmobilized conjugate having a specific affinity for human CK-MM. A moredirect and less expensive procedure, however, would be to immobilize theanti-CK-MM monoclonal antibody directly to the microbead support inaccordance with methods now well known in the art.

IV. Detecting Analytes

A. Using Color Change Chemistry to Detect Analytes

Any of a number of methods known to the art can be used to detect ananalyte collected on a patch of the present invention. One such methodinvolves the use of color change chemistry to visualize the presence ofan analyte on a patch. In this embodiment, after the test patch has beenworn for a suitable period of time, it can be removed by the wearer (innon-drug screen tests) and developed to produce a visible indicium ofthe test result. Such a test patch can be marketed together with adeveloper packet such as packet 34 shown in FIG. 4 which contains knowndeveloper reagents for the immunoassay. The reagent packet 34 comprisesa container 36 having a removably secured top 38. A flap 40 on the top38 of the reagent packet facilitates gripping the top 38 and peelingaway from container 36 to reveal the reagent contained therein. As anexample, a protein electrophoresis stain such as Coomassie brilliantblue or amido black 10b, can be bound to purified analyte contained inthe reagent packet 34. When a test patch is immersed in the packet 34,any antibodies on the test patch that are unbound by analyte in theperspiration will become occupied by stained purified analyte in thepacket 34. There will thus be an inverse relationship between the amountof stain absorbed by the patch and the amount of enzyme passed throughthe patch. In this embodiment, the user would place the patch in thefluid of the packet 34, wait for some period of time such as 30 secondsor more, rinse the patch under tap water and relate the resultant colorof the patch to the presence of the enzyme. A color comparison chart andcontrol zone on the patch having no bound antibody may be provided toaid in this interpretation.

Alternatively, the user could support the test patch on an open vessel,such as a small jar or vial, or empty container similar in design toreagent packet 34 securing the adhesive border of the patch to the rimof the vessel, and then pour contents of packet 34 on top of the testpatch. Gravity would assist the transport of the contents of packet 34through the test patch to maximize the efficiency of the stain/bindingreaction, and to facilitate visualization of the color change.

The system could readily be designed so that the user performs theinterpretation of the concentration of the analyte not in the patch atall but by observing the packet contents once the contents havetraversed the patch. This method would be similar to conventional ELISAassay methods where the packet contents contain enzyme conjugates whichwill react to specific enzyme substrates. The enzyme substrates would beadded to the packet contents after those contents transversed the testpatch.

If the perspiration contained molecules of interest, they would bind tothe specific immobilized binding partner on the patch. If this occurred,enzyme conjugates in the packet would pass freely across the test patchand enzymatically modify the enzyme substrate producing a controlledcolor change in the solution in the packet. If the perspirationcontained the desired molecules of interest, enzyme conjugates wouldthen be bound in transit across the patch and would be unavailable tocause color change in the substrate solution. Other immunoassay schemescan be readily adapted for use in the present invention by one of skillin the art.

A variety of well known immunoassay schemes for visualizing the presenceof an analyte of interest are well known in the art, and need not bedetailed here. However, the optimal immunoassay scheme is generally onewhich is simple and requires the fewest steps. For many types of assays,it will be desirable for the wearer to obtain rapid results such as acolor change to demonstrate a positive or negative result with as fewsteps as possible. On the other hand, drug of abuse screens are morelikely to be evaluated by clinical staff instead of by the test subject,and there is less concern for a "user friendly" product.

For example, in a patch of the present invention designed fordetermining both the presence of CK-MM and CK-MB enzyme, the immobilizedspecific binding partner for each of those enzymes will be segregated toseparate regions of the test patch. In this manner, if an enzyme-linkedimmunoassay system is utilized, a common enzyme and a common substratecould be used. Alternatively, a different color can be used to expressthe presence of different analytes.

B. Detecting a Metabolite of an Analyte Collected on a Patch

One problem which has been encountered in detecting analytes containedin patches, especially when such analytes are drugs of abuse, is thatmany conventional systems for performing drug testing do not test forthe analytes which are collected on a patch but rather for themetabolites of such analytes. This is because the analytes themselvesare not expressed in some body fluids. For example, cocaine is presentin perspiration but not in urine. Therefore, urine is not tested for thepresence of the cocaine molecule itself but rather for the presence ofthe major urine metabolite of cocaine in man, benzoylecgonine ("BE"), inorder to detect cocaine use by a subject.

Currently, the primary method for the diagnosis of drug abuse is byurine analysis. Many conventional diagnostic systems, therefore, aredesigned to screen for drug analytes (or their metabolites) in urine.For example, numerous companies have developed very sophisticatedautomated systems to quantify cocaine metabolites in urine. Such systemsare highly sensitive to the presence of the major cocaine metabolite inhuman urine, benzoylecgonine or BE. However, since the cocaine moleculeitself is not present in urine, many of these systems, such as the SYVAEMIT system (Palo Alto, Calif.) and Roche RIA system (Nutley, N.J.), arevirtually blind to the cocaine molecule itself.

In order to take advantage of conventional diagnostic systems thatperform drug abuse testing by urinalysis, it is important that the drugcontents of a patch of the present invention be measurable by suchdiagnostic systems. Unfortunately, most of the kits on the market whichtest for the presence of analytes such as cocaine are designed to detectmetabolites of such molecules rather than the analytes themselves. Inorder to utilize such diagnostic systems to test for a desired analyte,therefore, the contents of a patch must be chemically modified.

In accordance with another aspect of the present invention, therefore,an analyte contained in a patch which is not detectable by conventionaldiagnostic systems, particularly systems for performing urinalysis, ischemically modified so that it can be detected by such systems. In thisaspect, an analyte passed through the skin of a subject in perspirationis collected on an absorbent material in the patch. The analyte can thenbe chemically modified and detected while still in the absorbent layeror while bound to a microbead in a microbead layer. Alternatively, theanalyte can be freed from the absorbent material, such as throughchemical elution or by dissolving the absorbent material, in order toallow the analyte to be detected by a conventional diagnostic system.The analyte is then chemically modified so that it can be detected insuch a diagnostic system.

As long as the analyte and the metabolite of that analyte which isdetected by a diagnostic system are known and a means of converting theanalyte into its metabolite is known, it is within the knowledge of oneof skill in the art to chemically modify such an analyte so that it canbe detected. Thus, any such analyte contained in a patch of the presentinvention can be tested using conventional diagnostic systems. However,an example of how to chemically modify a particular analyte commonlytested for, cocaine, will be detailed below.

Cocaine is metabolized in the body by either pH changes orcholinesterase enzymes. Cocaine is unstable at pH values higher than 7,and thus can be converted to BE either through exposure to high pH or tocholinesterase enzymes. Therefore, in order to chemically modify thecocaine on a patch and convert it to BE in order to make it detectableby conventional urinalysis, cocaine molecules can be extracted from thepatch and then exposed to a solution at pH 11 at room temperature for 10minutes or more. Following this modification step, the patch extract isreturned to a neutral pH and then analyzed with conventional diagnosticsystems. As is obvious to one of skill in the art, other methods ofhydrolyzing the ester linkages of the cocaine molecule in order toproduce BE, such as through the use of enzymes, can also be performed inorder to prepare an extract of a patch of the present invention so thatit can be detected by conventional diagnostic systems.

C. Eluting Analytes from Dermal Patches

Another difficulty encountered in detecting analytes that are containedin perspiration and collected on a patch is that, unless color changechemistry is used to detect such analytes, these analytes usually haveto be removed from the patch in order to detect them. Removing theanalytes normally involves chemically eluting them from the patch, whichis both labor intensive and time consuming.

Therefore, in yet another aspect of the present invention, a patch isprovided in which the absorbent material of the patch on which analytesare collected is dissolvable. When such absorbent material is dissolved,the analytes contained therein are made available for detection throughfurther diagnostic procedures. As in other embodiments of a patch of thepresent invention, a patch incorporating a dissolvable absorbentmaterial is placed in fluid communication with the skin of a subject inorder to collect analytes contained in perspiration. Such a patch alsopreferably contains a gas permeable layer between the absorbent materialand the outside of the patch in order to allow the fluids expressedthrough the skin in perspiration to escape to the outside of the patchin their vapor phase.

The analytes of interest that are collected on the absorbent materialare preferably able to withstand the chemical treatment which results inthe dissolution of the absorbent material. Thus, the dissolution of theabsorbent material will not affect the analysis of the analytescontained in the absorbent material. One of skill in the art will beable to recognize whether a particular analyte will be chemicallychanged by a particular chemical treatment used to dissolve theabsorbent material. If one of skill in the art would be unsure as towhether a particular analyte would withstand such chemical treatment, itis a matter of routine experimentation to treat a sample of the analyteunder the conditions of the chemical treatment and then determinewhether the analyte has been chemically altered.

In another embodiment, the chemical treatment of the absorbent materialconverts an analyte of interest contained in the absorbent material intoa detectable metabolite or into some other detectable species. Forexample, the treatment of cocaine with a strong base converts it intoBE, a common cocaine metabolite found in urine. The same strong base canalso be used to dissolve an absorption disk made from a materialsensitive to strong bases. In this embodiment, the dissolving of theabsorbent material does not interfere with the analysis of the analytecontained in the absorbent material, but instead actually allows theanalyte to be analyzed.

An absorbent material for use in this aspect of the present inventioncan be made from any of a variety of materials which can be chemicallydissolved. For example, a number of materials are variously susceptibleto chemical attack and dissolution by acids and/or bases. Among thesematerials are Nylon 6/6 (sold as Vydyne 909 by Monsanto Co., St. Louis,Mo.), Phenolic (sold as Polychem 102 by Budd Co.), Polyester (PBT) (soldas Celanex 3300-2 by Celanese Plastics), and polyurethane (TP) (sold asPellethane 2363-55D by The Upjohn Co.). To dissolve any of thesematerials, an appropriately strong acid or base is added the material,as is known to those of skill in the art.

Absorbent materials can also comprise a woven protein web, such as a webmade from protein fibers approximately 0.03 inches thick. Such fibersare disclosed by Baumgartner, J. Forensic Sciences, 34:1433-1453 (1989).

Another dissolvable material which can be used as the absorbent materialin the patch of the present invention is polystyrene. In thisembodiment, solvents of polystyrene can be used to dissolve suchabsorbent material. Such solvents include chlorinated and aromatichydrocarbons, esters, ketones, essential oils of high terpene contentand turpentine. Specific examples of such solvents includecyclohexanone, dichloroethylene, and methylenedichloride.

Materials and solvents other than those listed above, of course, canalso be used in this aspect of the present invention. The foregoingmaterials and solvents are therefore exemplary of this aspect of thepresent invention and not intended to be limiting.

V. Quantitative Determination of an Analyte in Perspiration

A. Dermal Patches for the Quantitative Determination of an Analyte

In another aspect of the present invention, the amount of an analytethat is present in a given volume of a subject's perspiration can bediscovered. An embodiment of this aspect of the present invention isillustrated in FIG. 11. In this embodiment, a fluid permeable supportlayer 120 is in fluid communication with the skin 12 of a subjectmammal, such as a human, and is located between the skin 12 of thesubject and an absorptive layer 130 made of an absorptive material.

In the embodiment illustrated in FIG. 11, the support layer 120 alsocomprises a rate-limiting structure which limits the passage ofperspiration from the skin 12 to the absorptive layer 130 to a ratelower than the rate of insensible perspiration of the subject. Theinsensible rate of perspiration is the rate of continuous perspirationof a subject which occurs without regard to the regulation of thetemperature of the subject and which is not normally noticed by thatsubject. For humans, the rate of insensible perspiration of sweat glandsin the arm, leg or trunk is approximately 6-10 m¹ /m² /hr (Randall, W.C., Am. J. Phys. Med., 32:292 (1953)). Since the rate of perspiration ofthe subject will almost always. be equal to or greater than the rate ofpassage of such perspiration through the rate-limited support layer 120,the rate of perspiration passing into the absorptive layer 130 can bekept approximately constant.

The rate-limited support layer 120 can be made from any material whichcan control the rate of diffusion of the components of perspiration. Forexample, diffusion can be controlled by a membrane. The rate ofdiffusion of any particular membrane is related to physicalcharacteristics of the membrane such as its molecular composition,thickness, and, in the case of a porous type of membrane, its pore size.One example of a porous type of membrane which can be used as arate-limited structure in this embodiment of the present invention is apolyester-supported polycarbonate microporous membrane, such as thatmanufactured by Nuclepore (Menlo Park, Calif.). The pore density, poresize and thickness of the membrane can be adjusted to provide thenecessary limited fluid transport rate for this application. Anotherexample of a porous membrane is nylon 6,6, such as that manufactured byPall Corp. (Glencove, N.Y.).

An alternative to using a porous type rate-limited membrane is to use arate-limiting structure comprising a dialysis or osmotic non-porousmembrane. Such membranes have the advantage of having molecular weightspecificity, which may increase analyte sensitivity. For example, if onewere interested in collecting a therapeutic drug or its metabolites inthe absorptive layer 130 and these analytes had a molecular weight of1000 Dalton, one could choose a dialysis membrane which would pass onlymolecules which are smaller than 2000 Dalton in size. Larger moleculeswould be excluded from passing into the absorptive layer 130. Bylimiting the molecules which pass into the absorptive layer 130,interference by other components in perspiration in the laboratoryanalysis of the analyte in the absorptive layer 130 is minimized.Although the support layer 130 of this embodiment of the invention hasbeen described as comprising a rate-limited structure, one of skill inthe art will recognize that the support layer 130 and the rate-limitedstructure can be two separate membranes or structures in fluidcommunication with each other.

The absorptive layer 130 is located distally of the support layer 120 sothat said support layer 120 is between the subject's skin 12 and theabsorptive layer when the patch is being worn. The absorptive layer canbe made from any number of absorbent materials. If passive absorption ofan analyte is adequate to capture that analyte on the patch, then alayer of medical grade paper such as Filtration Sciences medical gradepaper (FS#39) will suffice. If active absorption is required thensubstances such as monoclonal antibodies specifically tailored for highaffinity to the analyte can be chemically coupled to the absorptivelayer 130 in order to concentrate the analyte on the absorptivematerial, as previously described.

In this embodiment of the invention, a gas permeable layer 140, which ina preferred embodiment is also an outer protective layer, is locateddistally from the skin 12 of the subject on the side of the absorptivelayer 130 opposite that which borders the support layer 120. The gaspermeable, outer protective layer 140 can be made, for example, from1625 Tegaderm wound dressing made by the 3M Company (St. Paul, Minn.).In a preferred embodiment, the gas permeable layer 140 extends beyondthe areas of skin 12 covered by the support layer 120 and the absorptivelayer 130 when the dermal patch 110 is applied to the skin 12 of asubject. In this way, the support layer 120 and absorptive layer 130 areprotected from external abrasion and wear.

A means for attaching the patch to the skin of a subject is alsopreferably applied to a portion of the outer protective layer 140 whichextends beyond the support layer 120 and the absorptive layer 130. Mostcommonly, the means for attaching is an adhesive composition. Forexample, in a patch 110 in which the outer protective layer 140(excluding that portion to which an adhesive is applied) isapproximately 14 cm², an adhesive can be applied to an area ofapproximately 1 cm around the outer perimeter of the outer protectivelayer 140 on the side of the outer protective layer 140 in contact withthe subject's skin in order to attach the patch 110 to the skin 12 of asubject.

In a more preferred embodiment, a pooling area 150 is formed between theouter protective layer 140 and the subject's skin 12 when the patch isworn on the subject's skin 12. Such a pooling area 150 can be formed,for example, by an area 152 of the outer protective layer 140 whichextends beyond the support layer 120 and the absorptive layer 130 and towhich no adhesive is applied. Such a pooling area 150 collects theexcess perspiration that is not diffused across the support layer 120and allows it to dissipate into the environment across the outerprotective layer 140. By providing such a pooling area, theback-diffusion of the components of perspiration across the skin 12 isminimized, since excess perspiration which is unable to pass into theabsorptive layer 130 is shunted into the pooling area 150. Since therate of flow of perspiration into the absorptive layer 130 is controlledby the rate-limiting structure of the support layer 120, the absorbentmaterial of the absorptive layer 130 is in fluid communication with thepooling area 150 only through the support layer 120.

This pooling area is unattached to the subject's skin, and provides asufficient amount of space to accommodate extra perspiration which doesnot pass across the rate-limited structure of the support layer 120. Forexample, during times of heavy exercise, the rate of perspiration of thesubject might rise well beyond the rate at which perspiration can bepassed into the absorptive layer 130. During such times of heavyperspiration, the pooling area 150 acts as a "shunt" to divertperspiration away from the support layer 120. The volatile components ofsuch perspiration then evaporate through the gas permeable layer 140. Inthis way, the back-diffusion of perspiration and the buildup of bacteriaunder the rate-limited structure of the support layer 120 can be avoidedor at least mitigated.

B. Using Dermal Patches to Determine the Amount of an Analyte inPerspiration

In order to determine the length of time a patch has been worn, theamount of a reference analyte contained in a certain volume ofperspiration of a subject must first be determined. This analyte must bepresent in an approximately constant amount in a given volume ofperspiration for the period of time that the patch is worn by a subject.Once such an analyte and its concentration in perspiration is known, theamount of time a patch is worn can be determined because the rate atwhich perspiration passes into the absorptive layer is heldapproximately constant by the rate-limited structure. Since the rate ofpassage of perspiration is known and the amount of the reference analytein a given volume is known, once the total amount of the analyte inabsorptive layer is known the amount of time the patch has been worn bya subject can be determined.

The volume of perspiration concentrated on a patch can also bedetermined through the use of this embodiment of the present invention.The rate-limited structure of the support layer 120 in this embodimentis preferably designed to allow the passage of perspiration to theabsorptive layer 130 at a rate lower than the minimal rate of passage ofperspiration through the skin, thereby assuring a relatively constantrate of flow of perspiration into the absorptive layer 130. The totalvolume of perspiration concentrated on the absorption disk is thusdirectly related to and can be determined by the duration of wear.

In order to quantitatively determine the amount of an analyte containedin a given volume of a subject's perspiration, a patch having arate-limited structure as described above is first placed on the skin ofa subject, preferably a mammal. Perspiration is then passed across thisrate-limited structure at a known rate. For example, if the rate atwhich perspiration is allowed to pass across the rate-limited structureis equal to or less than the insensible rate of perspiration of thesubject, perspiration will pass into the absorbent material atapproximately a constant rate. After a sufficient test period of timehas elapsed to allow a detectable amount of the analyte to be tested forto pass into the absorbent material, the patch is removed from the skinof the subject. When the patch is removed, the amount of time betweenthe placement of the patch on the skin of the subject and the removal ofthe patch is recorded.

In order to then determine the total volume of perspiration which haspassed into the absorptive layer 130 and concentrated analytes there,the rate of flow of perspiration into the absorptive layer 130 (asdetermined by the rate at which perspiration passes across therate-limited structure of the support layer 120) is first multiplied bythe amount of time the patch has been worn. This figure indicates thevolume of perspiration which has passed through the support layer 120and into the absorptive layer 130. The total quantity of analyte in theabsorptive layer is then determined. By dividing the total amount ofanalyte present by the total volume of perspiration which has passedinto the absorptive layer 130, the average amount of the analyte in agiven volume of the subject's perspiration can be determined.

The above described aspect of the present invention is thereby suited tobe used in many areas of diagnostics where quantitative informationabout a particular analyte is necessary. For example, this invention canbe used to monitor therapeutic drug administration, determine thenutritional adequacy of a subject's diet, or explore hormonal imbalancesin a particular subject.

VI. Prevention of Tampering with Dermal Patches

In some uses of the present dermal patches, it is advantageous toprovide a means for indicating whether a wearer has removed a patchduring the examination period, particularly in situations where a wearerhas an incentive to make sure that the patch produces a specific result.For example, if it is desired to determine whether a wearer has ingesteda drug of abuse, safeguards are desirably provided to prevent tamperingwith the dermal patch.

A. Dermal Patches with Radial Slits

One embodiment of a patch for preventing tampering is illustrated inFIG. 8. In this embodiment, the patch 62 is secured to the skin 64 withan adhesive member 65. The adhesive member 65 is preferably constructedof a material that is strong enough to hold the patch 62 to the skin 64,but that is relatively easily torn such as during removal of the patchfrom the skin. A suitable material for use in this preferred embodimentis Tegaderm 1625, manufactured by Minnesota, Mining, and ManufacturingCorp. of St. Paul, Minn. Other companies, including Avery and Johnson &Johnson, manufacture similar suitable materials; the Johnson & Johnsonproduct being sold under the trademark "Bioclusive." It has been found,however, that with sufficient patience, a wearer could remove anadhesive member of this type and replace it without leaving any visibleindication that the adhesive member has been removed. Therefore, in theparticularly preferred embodiment shown, the adhesive member 65 hasstress razors 66 in the form of a plurality of radial slits around itsouter perimeter. The stress razors 66 can be arranged in any of a widevariety of configurations and densities and accrue the advantage oftearing upon removal, as will be apparent to one of skill in the art.

In the embodiment illustrated in FIG. 8, the radial slits 66 extendapproximately 0.05 inches in length from the outer edge toward thecenter of the patch 62. The slits 66 may be arranged with any of avariety of regular or irregular spacings therebetween, and, in thepreferred embodiment are preferably spaced approximately every 0.10inches around the perimeter of the patch 62. The adhesive force of thematerial of the adhesive member 65 is preferably more than the forceneeded to tear the adhesive member at the stress razors 66, so that ifthe patch 62 is removed, the material of the adhesive member is torn.Thus, when a patch of this preferred embodiment is worn, a torn adhesivemember serves as an indication that the wearer has likely tampered withthe patch. Of course, the weakening of the adhesive member 65 may beaccomplished by providing perforations rather than slits and the slitsor perforations may be oriented in directions other than radially.

During storage prior to use, it is desirable to cover the adhesivemember to prevent it from sticking to any surface; otherwise the stressrazors 66 could become torn prior to use. Accordingly, in the preferredembodiment shown in FIG. 8, the patch is provided with an inner cover 69to protect the adhesive member 65. The inner cover 69 is removed toexpose the adhesive member 65 prior to application of the patch 62 to asubject's skin. Any of a variety of non-adherent materials known tothose of skill in the art may be used for the inner cover 69, such asthose commonly used to cover adhesive bandages.

The patch 62 is virtually impossible to remove and replace withoutshowing visible signs of tampering. Thus, any analytes in sweat producedfrom skin under the concentration zone 14 during the time the patch isworn should be present in the patch.

However, a particularly shrewd subject desiring to produce falsenegative results could obtain additional test patches. This shrewdsubject would obtain false negative results by removing the initiallyapplied test patch and replacing the test patch just prior to the timethe patch is to be removed for assay. In order to ensure that the patchremoved from the subject is the same patch which was initially appliedto the subject, an identifying marker which is difficult to reproducecan be incorporated into the patch. For example, a bar codeidentification strip 67, similar to the bar codes used at supermarketcheck out stands can be incorporated into the patch, preferably justbelow the adhesive member 65. For best results in protecting againstreplacement of the patch, it is important that the identifying markernot be easily removed and replaced without providing an indication thatthe patch has been tampered with.

In a preferred embodiment, the patch 62 has a filter 68 between theouter layer 65 and concentration zone 14, as described above inconnection with FIGS. 1-3a. In a particularly preferred embodiment, thefilter is a fluid permeable filter formed from a James River PaperDrape.

The preferred adhesive members of the embodiment shown in FIG. 8, madefrom adhesive materials, such as Tegaderm, which are relatively weak instrength, have generally been designed for hospital patients who are notexpected to perspire at high rates. Therefore, the moisture vaportransmission rate (MVTR) of these materials is relatively low. Forexample, the MVTR of Tegaderm is approximately 810 g/m² /day. However,an active person may perspire at instantaneous rates as high as 26000g/m² /day. Consequently, an active person may put out more sweat thanthese adhesive members can transmit to the atmosphere. If this sweataccumulates for any significant period of time, channels may be formedbetween the skin 64 and the adhesive member 65, allowing sweat to exitbetween the adhesive member and the skin, rather than be absorbed by thepatch 62.

B. Dermal Patches with Pinhole Perforations

In accordance with a further embodiment of the present invention forpreventing tampering, illustrated in FIG. 9, there is provided a patch70 having an adhesive member 72 which allows excessive sweat to befreely transmitted to the outside through pinhole perforations 73. Thepinhole perforations may be distributed throughout a wide band 75extending from the outer perimeter of the adhesive member to a narrowband 77 surrounding the test region 81 of the patch 70.

Sweat produced beneath test region 81, over which there are no pinholeperforations 73, will be absorbed by the test region and will not betransmitted to the outside. The test region 81 includes the area of thepatch 70 directly under the concentration zone 14 of the patch as wellas the area immediately outside this zone. The narrow band 77 outsidethe concentration zone 14 of the patch has no pinhole perforations 73,and substantially restricts sweat forming underneath the test region 81from communicating with the wide band 75 where sweat is transmitted tothe outside. The width of the narrow band 77, is preferably between0.025 and 0.250 inches, more preferably between 0.05 and 0.125 inches.Narrow band widths less than the preferred width are not expected tokeep contact with the skin, whereas narrow band widths greater than thepreferred width may allow sweat channels to form, creating a path forsweat forming within the test region 81 to communicate with the outside.

C Use of Soluble Markers to Prevent Tampering

A wearer of the patch in screenings for drugs of abuse would be expectedto be rather creative in circumventing the protections of the patch. Forexample, a creative wearer could try to wash out the concentrated sweatcomponents from the patch while the patch remains on the wearer's skin.Such washing could be attempted using a needle and syringe, such asthose commonly used by intravenous drug abusers for drug injection. Forthose patches employing specific binding chemistry, attempted elution ofthe concentrated components using water would likely prove unsuccessful.Even for those patches not employing specific binding chemistry for theanalyte being tested, elution with water alone would be difficult,requiring substantial volumes of water without triggering the detectionof tampering through the removal of the patch from the skin. However,certain analytes could successfully be at least partially eluted usingother solvents.

Thus, in order to detect tampering with the patch through elution of thepatch's contents using water or other solvents, a known amount of amarker which is readily soluble in either aqueous or non-aqueoussolvents, can be added to the concentration zone during manufacture ofthe patch. The marker should be easily quantifiable. The marker shouldalso be soluble in either aqueous or non-aqueous solvents depending onthe likely route of elution of the analyte. Additionally, the markershould be suitable for prolonged skin contact and not be readilyabsorbed by the skin. A variety of dyes used in the production of makeuphave these suitable characteristics. Oil red N (catalogue number29,849-2) sold by Aldrich Chemical Corp. of Milwaukee, Wis. is asuitable lipid soluble dye. DG01 red and DH60 yellow, both availablefrom Virginia Dare Extract Co. of Brooklyn, N.Y. are suitable watersoluble dyes. These water soluble dyes can be easily quantitated byelution from the patch followed by measuring optical density at 6500 nmfor the red or 5800 nm for the yellow dye. The quantity of dye remainingcan be compared with the range of the amount of dye found to beremaining in patches worn continuously without tampering for the samelength of time.

Non-visible markers could also be used to prevent the wearer of thepatch from obtaining feedback regarding the extent of marker remainingin the patch. A colorless protein could be used for this purpose. Aprotein should be chosen that is easily identified in the lab, and alsonot be expected in human sweat. For example, Bovine gamma globulins,such as those sold by Sigma Chemical Co. of St. Louis, Mo., could alsobe used as a marker. The presence of these markers can be easilyascertained using Bovine IgG RID kit, available from ICN of Costa Mesa,Calif.

Thus, when a suitable marker is employed within the patch, when thepatch is analyzed for the particular analyte being tested, the patch canalso be analyzed for the presence of the marker. For visible markers,such as makeup dyes, the presence of the marker may be analyzed bysimply viewing the patch. For non-visible markers, the non-visiblemarker can be assayed along with the analyte. A significant decrease inthe amount of marker present would be an indication of tampering throughelution of the patch with a solvent.

D. Use of Adulterant to Prevent Tampering

A further method of tampering with the patch would be to add anadulterant to the patch which interferes with the assay chemistry.Numerous materials have been used to adulterate urine tests for drugs ofabuse. The most commonly used, and generally most effective method ofproducing a false negative result in a urine test is to dilute the urineby ingestion of excessive amounts of fluids. Advantageously, thisapproach would not likely be successful in producing false negativeresults in the sweat collection patch of the present invention becauseinterstitial concentration of drug metabolites is less likely to beinfluenced by ingestion of fluids.

However, the addition of certain adulterants to the patch may interferewith the analysis chemistry. For example, acids and bases are known tointerfere with assays for many drug metabolites by altering themetabolites' molecular structure. Additionally, many household products,such as detergents, ammonia, ascorbic acid (Vitamin C), and drainopeners have been used to interfere with urine assays. These productsproduce extremes of pH or changes in other chemical parameters, andwould be expected to result in trauma to the skin if used in connectionwith tests using the patch of the present invention. This trauma couldbe noted by the technician removing the patch.

However, weak acids and bases, as well as eye drops sold under thetrademark "Visine," are also known to interfere with a variety of assaysfor drug metabolites in urinalysis. However, these materials would notbe expected to produce skin trauma. Thus, the use of these materials orother compounds interfering With an assay that do not cause skin traumamight go unnoticed by the technician removing the patch if the fluidcontents of the material have had time to evaporate across the outerlayer of the patch. However, "Visine" and most other adulterants wouldbe expected to contain ionic materials.

Thus, in order to detect the use of an adulterant, test strips can beincorporated into the patch which will detect the presence of variousionic materials or of extremes of pH. Litmus paper, such as Hydrion pHtest paper, available from Baxter Scientific Products, is well known asan indicator of variances of pH. Accordingly, a short piece, for example1 cm by 1/2 cm, of litmus paper could be incorporated into the patch todetect the various household products identified above which are knownto be highly acidic or basic.

Many test strips are also known for detecting the presence of ionicmaterials. For example Baxter Scientific Products supplies test stripsfrom a variety of manufacturers for the detection of each of thefollowing ions: aluminum, ammonium, chromate, cobalt, copper, ion,nickel, nitrate, peroxide, sulphite, tin, and calcium. In addition, teststrips sold under the name "Qantab" are available from Baxter ScientificProducts which identify the presence of chlorine ions. Other test stripsavailable from the same supplier show glucose, protein, and ketones.Most of these test strips are read by simply comparing the color of thestrips with a color chart included with the strips. Thus, the teststrips provide a simple method of identifying the introduction of any ofa variety of adulterant materials.

In order to detect adulterants, such as "Visine," which contain ionicmaterials not known to the person performing the test, the tester mustfirst assay the adulterant using a variety of test strips for ions toascertain which ions are present in the materials. Once the appropriateions are detected, the test strips corresponding to those ions can beincorporated into the patch in order to provide an indication that theadulterant has been added to the patch.

Curiously, any particular adulterant might produce false negativeresults in some assays and false positive results in others. For eachassay, the common adulterants which could be used to produce falsenegative results could be identified by testing the assays with theaddition of small amounts of these known materials. Test strips couldthen be included which would detect the addition of these adulterants.

In a preferred embodiment, the test strip or strips are placed facingthe skin, where the strips are not visible to the wearer. The wearer isthereby not provided any feedback which aids the wearer in deception.

E. Use of a Light Attenuation Layer to Prevent Tampering

Many biological compounds are known to be affected by various spectralbands of light energy. For example, urine samples for analysis of LSDmust be kept from exposure to strong light. (Schwartz, Arch. Inter. Med.148: 2407-12 (1988)). Further examples of compounds which requireprotection from light include cocaine hydrochloride (Martindale ExtraPharmacopoeia, 29th Ed., p. 1213) and morphine sulphate (Id., p. 1310).It is expected that these and other compounds may be affected byexposure to light while being concentrated in the collection patch aswell.

Many analytes to be determined by a patch of the present invention mayrequire collection and storage in the patch for prolonged periods oftime (up to several weeks). These analytes are, therefore, exposed tosubstantial quantities of photoradiation. This quantity ofphotoradiation may be substantially greater than during a urine assayfor the same or similar analyte. Also, many analytes have peculiarlyhigh sensitivity to light. Thus, for analytes of peculiarly highphotosensitivity or for those requiring prolonged collection andstorage, it is particularly important to shield photosensitive analytesfrom light during prolonged storage in the patch.

Accordingly, in still another embodiment of the present invention,illustrated in FIG. 10, there is provided a test patch 90 having a lightattenuation layer 92 between the outer adhesive layer 65 and theconcentration zone 14. In FIG. 10, the adhesive layer 65, is shownhaving stress razors 66, however, this feature is to be understood asbeing optional in this embodiment of the invention.

The attenuation layer 92 is provided in order to attenuate thetransmission of light into the concentration zone 14 where thebiological compound of interest is being collected and stored. The layer92 should be substantially impervious to the transmission ofphotoradiation, yet should also allow relatively unrestricted passage ofthe aqueous components of sweat to the outer adhesive layer 65. Thelayer 92 should be of sufficient porosity that diffusion of the aqueouscomponents of sweat occurs at least as rapidly as sweat normallyaccumulates in the patch.

Because light of many wavelengths is capable of degrading the variousbiological compounds which may be of interest, the layer 92 should haveoptical properties which attenuate light throughout a wide spectrum.Attenuation can be achieved by either reflection or absorption ofincoming light. Reflection may be achieved through, for example, the useof any of a variety of metallic surfaces. When used in accordance withcertain preferred embodiments of the present invention, the attenuationlayer 92 should allow passage of aqueous components of sweat. In orderto provide a reflective layer with the suitable permeability, thinmetallic foil with small holes can be provided. For example, aluminumfoil, commercially available from many sources including ReynoldsAluminum Co., could be perforated with a plurality of small holes.

Absorptive attenuation layers can be provided through the use of a blacksurface. Preferably, these surfaces would continue to allow permeabilityof aqueous components of sweat. It is important that any attenuationlayer on in the attenuation layer 92 not bleed when exposed to theaqueous components of sweat and also that it not interfere with anybinding chemistry or in the analysis of the analyte. Any of a variety ofthin black papers having these properties are commercially available andare suitable for use as in the attenuation layer. For example, blackDeltaware cellulose membrane filters available from Baxter ScientificProducts have been found to be especially useful for use as anattenuation layer. This product is available in a variety of porosities;more open pores are preferred. Thus, in the preferred embodiment, 0.6micron black Deltaware filters are provided.

In an alternative to the provision of an attenuation layer (not shown),the adhesive layer 65 can be made to attenuate light, either throughabsorption or reflection. As an example of an absorptive adhesive layer,black colorant, such as fine carbon black powder, could be incorporatedinto the extrusion of the adhesive sheet.

VII. Determining Allergic Sensitivity with a Dermal Patch

A. Dermal Allergic Reactions

In a further aspect of the present invention, a patch can be used todetermine whether a subject is allergic to a particular allergen.Allergens include various forms of pollen, dust, animal skin and fur,chemicals such as insecticides or food additives, and foods. Thepresence of an allergen on the skin of an individual sensitive to thatallergen causes an immune system reaction, known as an allergicreaction, in that individual.

Certain components of the immune system involved in provoking anallergic reaction, such as IgE, complement, and various immune cells,are believed to be able to migrate in the dermis. Components of theimmune system also circulate in the blood supplying the skin, and aspart of an allergic reaction to an allergen on the skin the permeabilityof the blood vessels supplying the skin is increased. Immune componentsof. the blood are thereby also believed to participate in a dermalallergic reaction. Thus, the presence of an allergen on the skin resultsin the migration and concentration of immune components of the body onthe surface of the skin where the allergen is present.

B. Using Dermal Patches to Determine Allergic Sensitivity

A subject, preferably a mammal, can be tested for its sensitivity to anallergen by contacting an allergen to the skin of the subject and thendetecting any immune components which pass through the skin of thesubject and onto a patch of the present invention. In this embodiment, apatch is used which contains an allergen in fluid communication with theskin of the subject when the patch is worn on the skin of the subject.For example, the allergen can be contained in the absorbent material ofthe patch.

In a preferred embodiment, an agent is also present in the patch influid communication with the skin of a wearer of the patch. The agent isone capable of increasing the permeability of the capillaries in thesubject's dermis. Such an agent can thus increase the permeability ofthe capillaries in the dermis beneath the patch and facilitate the flowof immune components to the site of the allergen.

To determine whether a subject is allergic to a particular allergen, apatch of the present invention which additionally includes an allergenis placed on the surface of the skin of the subject. In this embodiment,when perspiration reaches the patch, the allergen is in fluidcommunication with the skin of the subject and contacts the skin so asto cause an allergic reaction in the subject, if the subject issensitive to the allergen. The patch will then be able to collect bodilycomponents on the absorbent material of the patch which are associatedwith an allergic reaction, such as immune system components, whichmigrate to the location of the allergen. Once such components haveaccumulated in the absorbent material, the patch is removed, and thepresence of such components is detected. If such allergicreaction-associated components are present on the patch, this isindicative that the subject is allergic to the allergen tested.

Alternatively, the skin of the subject can be exposed to an allergen inany other way, such as simply by placing a sample of the allergen on theskin of the subject. Perspiration and other components expressed throughthe skin can then be accumulated in a patch of the present inventionlocated proximate to the area of the skin of the subject which wasexposed to the allergen. If an analyte indicative of an allergicreaction is then detected in the perspiration accumulated on the patch,the subject can be diagnosed as being allergic to the allergen.

The patch used in this embodiment of the present invention can be any ofthe types previously described. Preferably, a specific binding partnercapable of binding and concentrating particular bodily componentsindicative of an allergic reaction are included in the absorptive layer(or concentration zone) of this aspect of the present invention.

As an example of the present embodiment of the invention, an antigensuch as pollen can be placed in the absorptive layer of the patch sothat when perspiration penetrates the absorptive layer and bringsmoisture to that layer, the allergen can migrate through the absorptivelayer to the lower surface of the patch in contact with the skin andprovoke an allergic reaction, if the subject is prone to develop anallergic reaction to the allergen. Alternatively, the allergen can beplaced directly on the lower surface of the patch so that it immediatelycomes into contact with the skin of a subject wearing the patch.

After an immune response is triggered in a subject who is allergic tothe allergen, components involved in the response will increase inconcentration in the vicinity of the patch, since it is the site of theallergen. As sensible and insensible perspiration pass through the skinand into the patch, the immune components which pass through the skinwith such perspiration concentrate on the absorptive layer of the patch.

Agents which increase capillary permeability in the dermis immediatelybeneath the patch are preferably included in the patch. Moleculescirculating in the capillaries beneath the skin can thereby be made todiffuse into the interstitial space of the skin and from there intoperspiration. Such perspiration can then carry these molecules into thepatch so that they can be detected.

The following examples describe only specific applications of thepresent invention.

EXAMPLE 1 Preparation of Monoclonal Antibodies to CK-MB for Use on aTest Patch

In accordance with one known process for preparing monoclonalantibodies, mice such as Balb/c female mice or other mouse strains oreven other suitable animals such as rats or rabbits are immunized withan amount of the CK-MB enzyme to initiate an immune response. The enzymedosage and immunization schedule for producing useful quantities ofsuitable splenocytes can be readily determined depending on the animalstrain used.

The size and spacing of doses of CK-MB or other antigen are of primeimportance in the antibody response. Fortunately, a wide range ofantigen doses commonly affords immunity against harmful agents. Thus, asmall dose of antigen is usually sufficient to initiate an antibodyresponse, i.e., microgram quantities of proteins are frequentlyadequate. However, a minimum dosage for initiating an immune responsedoes typically exist, although doses of antigen below the minimum dosenecessary to initiate an antibody response will usually maintainantibody production which is already in process. For example, an initialimmunization with approximately 50 μg of the enzyme may be followed by ahyperimmunization series of five injections.

When certain compounds which are themselves not necessarily antigenicare mixed with an antigen, enhanced antibody production against theantigen occurs, as evidenced by the appearance of large amounts ofantibody in the serum, a prolonged period of antibody production, and aresponse to lower doses of antigen. Such substances are called"adjuvants" and include Freund's incomplete and complete adjuvants andalum gels. Thus, a given dose of antigen is usually more effective wheninjected subcutaneously with an adjuvant or when injected as repeatedsmall aliquots than when administered intravenously.

Typically, the adjuvants of Freund are preferred. The original"complete" Freund's adjuvant mixture consists of mineral oil, waxes andkilled tubercle bacilli. Antigen is added to the adjuvant mixture in anaqueous phase to form a water-in-oil emulsion in which each waterdroplet is surrounded by a continuous oil phase containing tuberclebacilli. The mixture is commonly injected subcutaneously intoexperimental animals. Injection stimulates a marked granulomatousreaction with lesions consisting largely of collections of histiocytes,epithelioid cells and lymphocytes. The local lymph node shows a smallincrease in plasma cells.

Following the immunization with a primary dose of a soluble proteinantigen, specific antibodies normally first appear in the serum after afew days and then increase in number until about the second week.Thereafter, the number of serum antibodies slowly declines over a periodof weeks to months.

The first serum antibodies to appear after antigenization are IgMantibodies. These are usually followed by the appearance of IgGantibodies. Later, as antibody serum levels increase, IgM antibodiesdisappear, probably as a result of specific feedback suppression of IgGantibodies.

After the "primary response" to a protein has passed, a second dose ofthe same antigen given months or even years later usually elicits anintense and accelerated "specific secondary response" in which serumantibody usually begins to rise within two or three days of exposure.The serum levels of antibody in a secondary response may reach as highas 10 mg per ml.

The animal is subsequently sacrificed and cells taken from its spleenare suspended in an appropriate medium and fused with myeloma cells,such as those obtainable from the murine cell line Sp2/O-Ag14. Theresult is hybrid cells, referred to as "hybridomas," which are capableof reproduction in vitro and which produce a mixture of antibodiesspecific to each of the various recognizable sites on the CK-MB enzyme.

The myeloma cell line selected should be compatible with the spleencells, and optimally should be a cell line of the same species as thespleen cells. Although the murine cell line Sp2/0-Ag14 has been found tobe effective for use with mouse spleen cells, other myeloma cell linescan alternatively be used. See, for example, Nature, 276: 269-270(1978).

The myeloma cell line used should preferably be of the so-called "drugresistant" type, so that any unfused myeloma cells will not survive in aselective medium, while hybrid cells will survive. A variety of drugresistant myelomas are known.

The mixture of unfused spleen cells, unfused myeloma cells and fusedcells are diluted and cultured in a selective medium which will notsupport the growth of the unfused myeloma cells for a time sufficient toallow death of all unfused cells. A drug resistant unfused myeloma cellline will not survive more than a few days in a selective medium such asHAT (hypoxanthine, aminopterin and thymidine). Hence, the unfusedmyeloma cells perish. Since the unfused spleen cells are nonmalignant,they have only a finite number of generations until they fail toreproduce. The fused cells, on the other hand, continue to reproducebecause they possess the malignant quality contributed by the myelomaparent and the enzyme necessary to survive in the selected mediumcontributed by the spleen cell parent.

The supernatant from each of a plurality of hybridoma containing wellsis evaluated for the presence of antibody to a specific site unique tothe CK-MB enzyme structure. Hybridomas are then selected producing thedesired antibody to that specific site. This selection may be, forexample, by limiting dilution, in which the volume of diluent isstatistically calculated to isolate a certain number of cells (e.g., 1to 4) in each separate well of a microliter plate. In this way,individual hybridomas may be isolated for further cloning.

Once the desired hybridoma has been selected, it can be injected intohost animals of the same species as those used to prepare the hybridoma,preferably syngeneic or semisyngeneic animals. Injection of thehybridoma will result in the formation of antibody producing tumors inthe host after a suitable incubation time, resulting in a very highconcentration of the desired antibody in the blood stream and in theperitoneal exudate of the host. Although the hosts have normalantibodies in their blood and exudate, the concentration of these normalantibodies is only about 5% of the concentration of the desiredmonoclonal antibody. The monoclonal antibody may then be isolated inaccordance with techniques known in the art.

EXAMPLE 2 Preparation of Microbead Test Patch

One specific application of the present invention is the dualdetermination of skeletal muscle and cardiac muscle status as a resultof exercise. A dermal patch is constructed in accordance with theembodiment illustrated at FIGS. 3 and 3a. The gauze layer is prepared bycutting a circular patch having an approximately 1-inch diameter from aJohnson & Johnson non-stick gauze pad. The inner and outer porous layersare next prepared by cutting two circular patches of Ultipor (nylon 6),from Pall Corporation in Glen Cove, N.Y. Ultipor membrane is both fluidpermeable and microporous, and a membrane is selected having, forexample, a 1 micron rating. The microbead layer is prepared bycovalently bonding monoclonal antibody raised against CK-MB to amultiplicity of polystyrene beads having a mean particle size of atleast about 10 microns.

The patch is assembled by distributing approximately 0.2 gram ofmicrobeads across the surface of one of the porous layers. The secondporous layer is thereafter disposed adjacent the microbeads, and thegauze layer is next placed on top of the second porous layer. At thispoint, the patch is upside-down. The peripheral edges of each of thefirst and second porous layers and the gauze layers are secured togetherby conventional heat-sealing techniques. Thereafter, the subassembly isturned over and an annular torus of adhesive tape having approximately a2-inch outside diameter and slightly less than a 1-inch inside diameteris secured thereto to produce a finished patch.

EXAMPLE 3 Cardiac Muscle Status Test

The patch of Example 2 is then secured to the chest of a healthy 40-yearold male and worn throughout a 36-mile (130-minute) bicycle ride. Uponremoval of the patch following the ride, the test patch is immersed in afirst solution containing an excess of enzyme labeled anti-CK-MB forapproximately 30 minutes, to permit conjugation of labeled antibody withimmobilized analyte. The patch is then rinsed under tap water to removeunbound labeled antibody and immersed in a second solution containing asubstrate for the bound enzyme label, which undergoes a color changewhen acted upon by the enzyme. Appearance of color through the topporous layer indicates the presence of CK-MB, and possible cardiacinjury. Comparison to a color chart permits rough quantification.

EXAMPLE 4 Test for Use of Marijuana

THC polyclonal antibody from sheep (available from Biogenesis,Bournmouth, England) is diluted 1:100 in PBS (pH 7.5). The antibodiesare bound to Gelman 0.45μ (SU-450) Ultrabind Supported Membrane,following the protocol in Gelman Original Equipment Manufacturerapplication P.N. 31,084. The membranes are air dried. Disks, 3/8 inch indiameter, are cut from the coated Gelman membrances. The 3/8 inch disksare mounted at the center of a 1/4 inch diameter hole cut in the centerof a one inch diameter circle of Tegaderm 1625 Transparent Dressing(available from Minnesota Mining and Manufacturing, St. Paul, Minn.).

Three mounted membranes are secured to the chest of a subject who thensmokes a marijuana cigarette. Three mounted membranes are also securedto a subject who has never used marijuana in any form and who agrees notto use it for the next seven days. The membranes remain in place untilthey are removed, seven days later. Each of the removed membranes isflushed five times with 300 μl of 0.2% Tween 20 in PBS. The membranesare incubated for 30 minutes in 100 μl of E-Z Screen Cannabinoid enzymeconjugate from the E-Z Screen Test Kit (available from EnvironmentalDiagnostics, Inc., Burlington, N.C.).

After incubation, each membrane is flushed three times with 300 μl of0.2% Tween 20 in PBS, followed by three flushes with PBS alone. Themembranes are then incubated in TMB Membrane Peroxide Substrate(available from Kirkegaard & Perry Labs, Gaithersburg, Md.) for 10minutes. A light blue background appears in all six membranes. Whitedots appear over the background on the three membranes taken from thesubject who smoked a marijuana cigarette, indicating sweat gland outputof sweat containing THC derivatives. No white dots appear on the threemembranes taken from the subject who has never used marijuana.

EXAMPLE 5 Positive Control Patch

Mouse anti-human IgG, Fc monoclonal antibody (available from ICN, CostaMesa, Calif.) is diluted 1:100 in PBS (pH 7.5). The antibodies are boundto Gelman 0.45μ (SU-450) Ultrabind Supported Membrane, following theprotocol in Gelman Original Equipment Manufacturer application P.N.31,084. The membranes are air dried. Disks, 3/8 inch in diameter, arecut from the coated Gelman membranes. These 3/8 inch disks are centeredand mounted on a 1/4 inch diameter hole cut in the center of a one inchdiameter circle of Tegaderm 1625 Transparent Dressing.

Three mounted membranes are secured to the chest of five human subjects.The membranes remain in place until they are removed, seven days later.Each of the removed membranes is flushed five times with 300 μl of 0.2%Tween 20 in PBS. The membranes are incubated for 30 minutes in 100 μl ofHorseradish peroxidase enzyme conjugated to goat anti-human IgG, Fcpolyclonal antibody (available from ICN, Costa Mesa, Calif.) diluted1:1000 in PBS.

After incubation, each membrane is flushed three times with 300 μl of0.2% Tween 20 in PBS, followed by three flushes with PBS alone. Themembranes are then incubated in TMB Membrane Peroxide Substrate(available from Kirkegaard & Perry Labs, Gaithersburg, Md.) for 10minutes. Blue dots corresponding to individual sweat ducts appear overthe background on all of the membranes, indicating that the chemistry ofthe patches is operative by their detection of the IgG expected in thesweat of all subjects.

EXAMPLE 6 Chemical Modification of Cocaine Collected on a Patch

Absorption disks, 3/8 inch in diameter, are cut from Gelman membranes(Gelman 0.45μ (SU-450) Ultrabind Supported Membranes). These 3/8 inchdisks are mounted at the center of a 1/4 inch diameter hole cut in thecenter of a one inch diameter circle of Tegaderm 1625 TransparentDressing (available from Minnesota Mining and Manufacturing, St. Paul,Minn.) to form a patch.

Three of such patches are secured to the chest of a subject who theningests cocaine. Three patches are also secured to a subject who hasnever used cocaine in any form and who agrees not to use it for the nextseven days. The patches remain in place until they are removed sevendays later from each subject.

The cocaine molecules and other components present in the membranes ofeach patch are then eluted from the membranes by soaking each of themembranes in a synthetic urine matrix for 30 to 60 minutes at roomtemperature with mechanical agitation to form an analyte solution.Following elution, the analyte solutions derived from each of thepatches are brought to a pH of 11 by the addition of NaOH to each of thesolutions. The solutions are reacted for 20 minutes at pH 11 and at roomtemperature, after which the solutions are neutralized with HCl.

Each solution is then subjected to diagnostic analysis with the RocheRIA system (Nutley, N.J.) for detecting the metabolite of cocaine BE.The subject who ingested cocaine tests positive for the cocainemetabolite BE, while the subject who did not consume cocaine over thetest period does not test positive for BE.

EXAMPLE 7 Preparation and Use of a Dissolvable Absorption Disk

Nylon 6/6 fibers (Vydyne 909 from Monsanto Co.) are formed into anabsorbent gauze. Disks approximately 3/8 inch in diameter are cut fromsuch gauze and are then mounted at the center of a 1/4 inch diameterhole cut in the center of a one inch diameter circle of Tegaderm 1625Transparent Dressing (available from Minnesota Mining and Manufacturing,St. Paul, Minn.) to form a patch. Such a patch is then applied to asubject. The subject is directed to ingest cocaine, and a quantity ofperspiration is then allowed to accumulate on the patch.

When a sufficient period of time has passed for a detectable amount ofcocaine to accumulate on the patch, the patch is removed from thesubject and placed in an insoluble container. A base capable ofdissolving the Nylon 6/6 fibers is then poured over the patch. Once thenylon absorption disk is dissolved, the undissolved components of thepatch are removed from the container. Since cocaine is converted intobenzoylecgonine (BE) in the presence of a base, the cocaine contained inthe disk is metabolized to BE when the disk is dissolved.

The solution of the dissolved nylon, BE, and the other remainingcomponents of the used absorption disk are next neutralized. Thissolution is then analyzed using a Roche RIA system (Nutley, N.J.). TheBE in the solution is detected and the amount of BE concentrated in theabsorption disk is determined.

EXAMPLE 8 Quantitative Determination of a Component of Perspiration

To determine how much of an analyte is contained in a given volume ofsweat, a patch is first constructed having a support layer made from apolyester-supported polycarbonate microporous membrane, manufactured byNuclepore (Menlo Park, Calif.). Over this is placed an absorbentmaterial such as Filtration Sciences medical grade paper (FS#39) foraccumulating and concentrating perspiration. The surface area of thelayer of absorbent material should be the same as or smaller than thatof the support layer so that when placed on a subject's skin, only thesupport layer is in contact with the subject's skin. Over this layer isthen placed an outer protective layer made of 1625 Tegaderm wounddressing made by the 3M Company (St. Paul, Minn.). This outer layer isof a larger surface area than either the support layer or the absorbentmaterial and covers both of these layers. The outer layer separates theabsorbent material from the outside of the patch and helps preventperspiration from entering the absorbent layer except through thesupport layer. The outer perimeter of the outer layer has an adhesive onthe side of the outer layer that faces the skin of a subject when thepatch is applied to the skin of such a subject in order to secure thepatch.

Such a patch is next placed on the skin of a subject whose perspirationis to be tested for the presence of theophylline. The subject wears thepatch for 7 days, during which time perspiration passes through thesupport layer at a rate of less than 6 grams/m² /hour. After this thepatch is removed and subjected to analysis to determine the amount oftheophylline contained in the patch.

To determine the volume of sweat that has passed into the absorbentmaterial of the patch, the rate at which perspiration passed into theabsorbent material is multiplied by the amount of time the patch wasworn, i.e., 7 days. The amount of theophylline contained in the patch isthen determined. These numbers are then related in order to determinethe amount of analyte contained in a given volume of perspiration bydividing the amount of the analyte in the patch by the volume ofperspiration which passed through the support layer into the absorbentmaterial.

EXAMPLE 9 Preparation and Use of a Dermal Patch to Determine theSensitivity of a Subject to an Allergen

In order to determine whether an individual is allergic to cat hair, apreparation containing cat hair is first placed on the lower surface ofa disk 3/8 inch in diameter made of Filtration Sciences medical gradepaper (FS#39). The upper surface of the disk is mounted at the center ofa 1/4 inch diameter hole cut in the center of a one inch diameter circleof Tegaderm 1625 Transparent Dressing (available from Minnesota Miningand Manufacturing, St. Paul, Minn.). The patch is then placed on thesurface of the skin of a human subject for approximately 3 days in orderto accumulate perspiration on the disk and form a concentrate. The diskis then removed and analyzed to detect IgA against cat hair. Thepresence of IgA against cat hair indicates that the subject hasexpressed an allergic reaction to the cat hair antigen.

Although this invention has been described in terms of certain preferredembodiments and assay schemes, other embodiments and assays that areapparent to those of ordinary skill in the art are also within the scopeof this invention. Accordingly, the scope of the invention is intendedto be defined only by reference to the appended claims.

What is claimed is:
 1. A dermal patch to be worn on the skin of a subject mammal for quantitatively determining the presence and amount of an analyte in said subject's perspiration, comprising:a fluid permeable support layer having a first and second side, and being adapted to be in fluid communication with the subject's skin when said first side is proximate to the subject's skin, wherein said support layer comprises a rate-limited structure that limits the rate of diffusion of perspiration through said support layer; and an absorbent material located adjacent to said second side of said support layer and in fluid communication with said support layer for collecting non-aqueous components of perspiration which diffuse through said support layer.
 2. The patch of claim 1, wherein said patch additionally comprises a gas permeable outer layer having a first and second side and an outer perimeter, said first side of the permeable outer layer being adjacent to said absorbent material and spaced apart from said second side of said support layer, wherein water and other fluids expressed through the skin of said subject are permitted to escape from said absorbent material through said gas permeable outer layer in their vapor phase.
 3. The patch of claim 2, further comprising a pooling area located proximate to said first side of said gas permeable outer layer and formed by a portion of said gas permeable outer layer that is unattached to the subject's skin when said first side of said support layer is proximate to the subject's skin for collecting excess perspiration that is not diffused across said support layer, wherein said absorbent material is in fluid communication with said pooling area only through said support layer.
 4. The patch of claim 2, wherein said gas permeable outer layer further includes an adhesive composition applied to the outer perimeter of said gas permeable outer layer on the first side of said gas permeable outer layer, said adhesive composition for attaching said patch to the skin of said subject.
 5. The patch of claim 1, wherein said rate-limited structure is selected from the group consisting of a polycarbonate microporous membrane and a membrane made from nylon 6/6.
 6. The patch of claim 1, wherein said absorbent material is paper.
 7. The patch of claim 1, wherein said rate-limited structure limits the rate of diffusion of perspiration across said support layer to less than the insensible rate of perspiration through the skin of said subject.
 8. The patch of claim 1, wherein said absorbent material is provided with a specific binding partner for an analyte present in the perspiration of said subject.
 9. The patch of claim 8, wherein said binding partner is an antibody.
 10. The patch of claim 1, wherein said absorbent material can be dissolved into a solution such that the dissolved material and solution do not interfere with the analysis of said analyte.
 11. A method of quantitatively determining the presence of an analyte contained in the perspiration of a subject mammal, comprising the steps of:a. placing a patch on the skin of a mammal, wherein said patch comprises an absorbent material capable of concentrating non-aqueous components of the perspiration of said mammal and a rate-limited structure having a known area, wherein said rate-limited structure is positioned adjacent to said absorbent material and proximate to the skin of said mammal when said patch is placed on the skin; b. passing perspiration of said mammal containing an analyte through said structure at a known rate to said absorbent material; c. removing said patch after a sufficient test period of time has elapsed so that said analyte can be detected by an assay for said analyte; d. recording the amount of time the patch was worn in order to determine the total amount of perspiration which passed across said structure; e. determining the amount of said analyte contained in the patch; and f. relating the amount of analyte determined in step (e) to the amount of perspiration determined in step (d) in order to determine the average amount of the analyte in said mammal's perspiration.
 12. The method of claim 11, wherein said structure is selected from the group consisting of a polycarbonate microporous membrane and a membrane made from nylon 6/6.
 13. The method of claim 11, wherein said absorbent material is paper.
 14. The method of claim 13, wherein a binding partner for said analyte is attached to said absorbent material.
 15. The method of claim 14, wherein said binding partner is an antibody. 